Isolated human EDG-4 receptor and polynucletide encoding said receptor

ABSTRACT

A lysolipid receptor, a human EDG-4 receptor, a method of identifying lysolipid receptors involved in inflammatory response and the lysolipid receptors so identified, and a method of identifying ligands which interact with such lysolipid receptors.

This application is a 371 of WO 99/35259 filed on Dec. 30, 1998, whichclaims priority from provisioned application No. 60/070185, filed Dec.30, 1997 which claims priority from provisioned application No.60/080,610 filed Apr. 3, 1998 which claims priority from provisionalapplication No. 60/109,885 filed Nov. 25, 1998.

FIELD OF THE INVENTION

The present invention is in the field of molecular biology. Moreparticularly, the present invention relates to a novel lysolipidreceptor, a human EDG-4 receptor, a method of identifying lysolipidreceptors involved in inflammatory response and the lysolipid receptorsso identified, and a method of identifying ligands which interact withsuch lysolipid receptors.

BACKGROUND OF THE INVENTION

(a) EDG Receptors

EDG receptors have been grouped with orphan receptors because theirendogenous ligands are not known (for example see Hla T and Maciag T(1990) J Biol. Chem. 265:93018-13; U.S. Pat. No. 5,585,476). Recently,however, lysophosphatidic acid (LPA) has been demonstrated to be theendogenous ligand for the EDG-2 receptor (Hecht et al. (1996) J. Cell.Biol. 135: 1071-1083; An et al. (1997) Biochem. Biophys. Res. Comm. 213:619-622).

The EDG receptors are seven transmembrane G protein coupled receptors(T7Gs or GPCRs). GPCRs are so named because of their seven hydrophobicdomains of 20-30 amino acids which span the plasma membrane and form abundle of antiparallel α helices. These transmembrane segments (TMS) aredesignated by roman numerals I-VII and account for structural andfunctional features of the receptor. In most cases, the bundle ofhelices forms a binding pocket; however, when the binding site mustaccommodate more bulky molecules, the extracellular N-terminal segmentor one or more of the three extracellular loops participate in bindingand in subsequent induction of conformational change in intracellularportions of the receptor. The activated receptor, in turn, interactswith an intracellular G-protein complex which mediates furtherintracellular signaling activities such as the production of secondmessengers such as cyclic AMP (cAMP), phospholipase C, inositoltriphosphate, activation of protein kinases, alteration in theexpression of specific genes.

When the receptor is activated by the binding of a ligand, theconformation of the receptor changes allowing it to interact with andactivate a G protein. The activated G protein causes a molecule ofguanosine diphoshate (GDP), that is bound to the surface of the Gprotein, to be replaced with a molecule of guanosine triphosphate (GTP),which causes another alteration in the conformation of the G protein.With GTP bound to its surface the G protein can regulate the activity ofan effector. These effectors include enzymes such as adenylyl cyclaseand phospholipase C and certain transport protein and ion channels suchas calcium ions, potassium ions or sodium ions.

GPCRs are expressed and activated during numerous developmental anddisease processes. Identification of a novel GPCR provides theopportunity to diagnose or intervene in such processes. The receptor canbe used in screening assays to identify physiological or pharmaceuticalmolecules which trigger, prolong or inhibit a receptor's activity or adifferentially modulate distinct intracellular pathways which arecontrolled by GPCRs. However, for many of the GPCRs (such as the EDGreceptors) the biological processes mediated by the receptor arecurrently unknown. There exists a need therefore for methods to identifythe biological processes mediated by these GPCRs and also for methods ofidentifying other GPCRs that may be involved in these processes.

Because there are diverse functions of GPCRs, it is not surprising thatthere are a number of therapeutic drugs that act by modifying thefunction of GPCRs. Therapeutic drugs which modify the GPCRs areparticularly attractive because of the ability to design such drugs withparticular specificity so that they turn on or off specific receptorsand their signaling pathways.

(b) Lysophogpholinids and Inflammation

LPA is a naturally-occurring agonist of the EDG-2 receptor (Hecht et al.J Cell Biol 135: 1071, 1996). LPA, and many other lysophospholipids, areproduced by activated platelets as a consequence of inflammation-relatedintracellular signal transduction accompanying aggregation and thrombusformation. Similar inflammatory pathways occur in many cell types, andtypically lead to production of LL and other lipid mediators withinseconds to minutes, and activation of new gene expression within minutesto hours.

A number of lysop pholipids have been studied to determine theirbiological effects. For example, the lysophospholipdsphingosine-1-phosphate (S1P) appears to play a role in a number ofCNS-related biological processes. These include apoptosis, mitogenesisand cytoskeletal reorganization. S1P has been proposed to mediate atleast some of the biological functions of PDGF and NGF. The former is agrowth hormone with potent mitogenic and wound-healing activity. Thelatter is a neurotrophic factor, which has also been proposed to play arole in neuropathic pain.

In addition, it has been reported that there is activation of NF-κB byS1P in U937 cells; however, the authors assumed that S1P was anintracellular second messenger, and no attempt was made to determinewhether this response was receptor-mediated. Furthermore, the functionalrelevance of NF-κB activation was not tested, e.g. by examining thepossible upregulation of inflammatory cytokines, adhesion molecules orother NF-κB-dependent genes. If multiple receptors for S1P exist, thefinding of NF-κB activation offers no utility by itself, since one,several, or all of the receptors might respond through NF-κB.

Moreover, direct modulation of NF-κB activation cascades has beenproposed as a therapeutic mechanism for inflammation or apoptosis.However, NF-κB plays a vital role in innate immunity against ubiquitousmicrobial pathogens and in mobilizing the antigen-specific immunesystem. Therefore, rather than targeting this irreplaceable defensesystem, it would be preferred to instead block inappropriate activationof NF-κB by specific inflammatory or apoptotic signaling events.Accordingly, it is highly desireable to design therapeutic agents whichcould modulate NF-κB activation and thereby prevent unwanted apoptosisor thereby enhance immune function in immunocompromised hosts via areceptor modulated pathway.

SUMMARY OF THE INVENTION

It has now been discovered that there are LL/EDG receptors which areinvolved in an inflammatory response signaling pathway and an apoptoticsignaling pathway. In particular, it has been discovered that the EDG-2,EDG-3, EDG-4, EDG-5 and EDG-6 receptors activate NF-κB and/or theproduction of IL-8. Accordingly, the present invention provides a linkbetween NF-κB activation and edg receptors and hence a means forcontrolling NF-κB activation and thereby for controlling apoptosis andinflammatory responses.

In an aspect of the present invention, it has been discovered thatagonists to the EDG-2, EDG-5 and EDG-6 receptors result inactivation/production of NF-κB and/or IL-8. In particular, it has beendiscovered that LPA will act as an agonist to the EDG-2, EDG-5 and EDG-6receptors resulting in activation/production of NF-κB and/or IL-8.

In another aspect of the present invention, it has been discovered thatagonists to the EDG-3 and EDG-4 receptors result inactivation/production of NF-κB and/or IL-8. In particular, it has beendiscovered that S1P and SPC will act as an agonist to the EDG-3 andEDG-4 receptor resulting in activation/production of NF-κB and/or IL-8.

In another aspect of the present invention there is provided isolatedpolynucleotides encoding the human EDG-4 receptor. The isolatedpolynucleotides may be either cDNA or genomic clones.

In particular, the present invention provides an isolated nucleotidesequence selected from the group consisting of:

a) the nucleotide sequence comprising nucleotides 38-1099 of FIG. 15A(SEQ ID NO:1);

(b) the nucleotide sequence of FIG. 15B (SEQ ID NO:2);

(c) a nucleotide sequence with at least about 95% sequence identity to(a) or (b) and which hybridizes under stringent conditions to sequences(a) and (b), respectively;

(d) a nucleotide sequence which encodes the amino acid sequence of FIG.16A (SEQ ID NO:3) for the human EDG-4 receptor; and

(e) a nucleotide sequence which encodes the amino acid sequence of FIG.16B (SEQ ID NO:4) for the human EDG-4 receptor.

There is also provided: expression vectors; host cells; purified aminoacid sequences; complementary nucleic acid sequences; biologicallyactive fragments; and hybridization probes, for such nucleotidesequences and their encoded amino acid sequences.

In another aspect of the present invention, there is provided a methodof determining whether a DNA sequence encodes edg receptors that areinvolved in inflammatory response by measuring the induction of NF-κBand/or IL-8 upon activation by a suitable ligand.

In another aspect of the present invention, there is provided a methodof determining whether a DNA sequence encodes an edlfosine receptor thatis involved in inflammatory response by measuring the induction of NF-κBand/or IL-8 activation by a suitable ligand, including edelfosine.

In another aspect of the present invention, there is provided a methodof identifying ligands that interact with edg or lysolipid receptorsthat are involved in inflammatory response. In particular, the presentinvention provides a method of identifying ligands which interact withedg or lysolipid receptors by measuring the induction or lack ofinduction of NF-κB and/or IL-8.

In another aspect of the present invention, there is provided a methodof modulating or treating an inflammatory process condition in a subjectby administering an effective amount of a pharmaceutical compositioncomprising an agonist or antagonist of an NF-κB and/or IL-8 modulatedEDG or lysolipid receptor and a pharmaceutically acceptable excipient,for upregulation or downregulation of the inflammatory process,respectively. In particular, agonists and antagonists of the EDG-2,EDG-3, EDG-4, EDG-5 and/or EDG-6 receptor are applicable.

In another aspect of the present invention, there is provided a methodof modulating an immune response in a subject by administering aneffective amount of a pharmaceutical composition comprising an agonistor antagonist of an NF-κB and/or IL-8 modulated EDG or lysolipidreceptor and a pharmaceutically acceptable excipient, for upregulationor downregulation of the immune response, respectively. In particular,agonists and antagonists of the EDG-2, EDG-3, EDG-4, EDG-5 and/or EDG-6receptor are applicable.

In another aspect of the present invention, there is provided a methodof controlling apoptosis by activating an EDG or lysolipid receptorwhich receptor activates the induction of NF-κB. In particular, bymodulating the EDG-2, EDG-3, EDG-4, EDG-5 and/or EDG-6 receptor viaagonists or antagonists there is provided a method of controllingapoptosis.

An EDG receptor herein refers to any receptor with at least 27-30%identity, preferably at least 30-35% identity, more preferably at least35-40% identity, even more preferably at least 40-45% and mostpreferably at least 45-50% identity with each other. As is known in theart, the percentage identity of the amino acid sequences of relatedreceptors is generally greater in the same species than in differentspecies.

BRIEF DESCRIPTION OF THE FIGURES

The following figures will now be used to describe the invention in moredetail.

FIG. 1A illustrates the chemical structure of LPA, S1P, SPC andpyschosine.

FIG. 1B illustrates the time- and concentration-dependent IL-8 responseto S1P and LPA in HeLa cells.

FIG. 2A illustrates the concentration dependent IL-8 response to S1P andSPC in HeLa cells.

FIG. 2B illustrates the concentration-dependent IL-8 response to S1P andSPC in HeLa cells and the PTX-sensitivity of this response.

FIG. 3 illustrates the IL-8 response to S1P and TNF-α in HeLa cells andthe PTX- and genistein sensitivity of this response.

FIG. 4A illustrates the concentration-dependent IL-8 response to S1P,sphingosine and sphingomyelin in HeLa cells.

FIG. 4B illustrates the IL-8 response to lysolipids in primary culturedHuman Umbilical Vein Endothelial Cells (HUVEC).

FIG. 5 illustrates the time- and concentration-dependent IL-8 responseto TNF-α, S1P and LPA in HL-60 cells.

FIG. 6 illustrates the concentration-dependent IL-8 response to S1P inHeLa and HL-60 cells, as well as the cell viability at each S1Pconcentration level.

FIG. 7 illustrates the effect of surarmin on the IL-8 response to S1P inHeLa cells.

FIG. 8 illustrates the effect the antioxidants NDGA and NAC on the IL-8response to S1P in HeLa cells.

FIG. 9 illustrates the IL-8 response to edelfosine in HeLa cells and thePTX- and suramin sensitivity of this response.

FIG. 10A illustrates the IL-8 response to S1P in 293-EBNA cellstransfected with rat EDG-4 expression plasmid and the PTX sensitivity ofthis response.

FIG. 10B illustrates the expression of endogenous edg receptors in HeLa,COS and 293-EBNA cells.

FIG. 11 illustrates the NF-κB reporter response to S1P, LPA and SPC in293-EBNA cells cotransfected with an edg4 expression plasmid and aNF-κB-tk-p4Luciferase reporter plasmid.

FIG. 12 illustrates the NF-κB reporter response to S1P, LPA, pyschosine,SPC, LPC, sphingosine, 20% FBS, TPA, lysosulfatide and edelfosine in293-EBNA cells cotransfected with an EDG-4 expression plasmid and anNF-κB-tk-p4Luciferase reporter plasmid, as well as the PTX sensitivityof this response.

FIG. 13 illustrates the EDG-1, EDG-3 and EDG-4 receptor response to S1Por SPC using (A) the SRE reporter gene assay or (B) theNF-κB-tk-p4Luciferase reporter assay.

FIG. 14 shows a multiple alignment of EST sequences representing the 5′end of the open reading frame of human EDG-4 cDNA. Sequences werealigned using the PILEUP program from the Wisconsin Package Version 9.0,Genetics Computer Group (GCG), Madison, Wis. The predicted translationstart of human EDG-4, based on similarity to the rat translation startsite, begins at nt 45 of the multiple alignment.

FIG. 15A, SEQ ID NO: 1, shows human EDG-4 cDNA and EDG-4 predicted aminoacid sequence. The cDNA sequence was derived from clones pC3-hedg4#5 andpC3-hedg4#36 isolated by PCR from human lung fibroblast cell line WI-38cDNA library (Origene Technologies Inc.).

FIG. 15B, SEQ ID NO: 2, shows human EDG-4 cDNA of clone pC3-Hedg4#36.

FIG. 16A, SEQ ID NO: 3, shows the amino acid sequence and features ofthe predicted polypeptide product of human EDG-4 cDNA of FIG. 15A.

FIG. 16B, SEQ ID NO: 4, shows the amino acid sequence of the EDG-4polypeptide encoded by pC3-hEdg-4#36.

FIG. 17A shows the GAP alignment of the predicted human vs rat EDG-4polypeptides. The predicted amino acid sequences of two polypeptideswere aligned using the GCG GAP program.

FIG. 17B shows the alignment of the amino acid sequences of EDG-4 asderived from the clones pC3-Hedg4#5 and pC3-Hedg4#36 (FIG. 16A) withpC3-Hedg4#36 (FIG. 16B) and with rat EDG-4/H218 using the PILEUPprogram.

FIG. 18A illustrates the SRE reporter response to SPC in 293-EBNA cellscotransfected with a human or rat edg4 expression plasmid and an SREreporter plasmid.

FIG. 18B illustrates the concentration-dependence of SRE response to S1Panalogs in EDG-4 transfected cells.

FIG. 19 illustrates the incellular calcium response to S1P in cellstransfected with the empty expression vector pcDNA3.

FIG. 20 illustrates the intracellular calcium response to S1P in cellstransfected with human EDG-3 expression vector.

FIG. 21 illustrates the amino acid sequence for human EDG-6 receptor.

FIG. 22 illustrates the cDNA sequence for human EDG-6 receptor.

FIG. 23 illustrates that the three LPA receptor subtypes signal throughNF-B and AP-1 genes.

FIG. 24 illustrates the SRE Response for a human EDG-4 fusion proteinwith Jellyfish Green Fluorescent Protein (GFP).

FIG. 25 illustrates edg receptors implicated in the activation of NF-κB.

DETAILED DESCRIPTION OF THE INVENTION

The EDG receptors are characterized by structural features common to theG-protein coupled receptor class, including seven transmembrane regions,and by the functional properties of binding lysophospholipids orlysophingolipids selectively. When expressed functionally in a hostcell, i.e., in operable linkage with a responsive second messengersystem the EDG receptors are capable further of responding tolysophingolipid or binding by signal transduction.

In the present invention it has been discovered that EDG receptors areinvolved in an inflammatory response signaling pathway and an apoptoticsignaling pathway by the activation of NF-κB and production of IL-8.

It has also been discovered that endogenous LL receptors in HeLa cellscan be activated to induce NF-κB/IL-8 and that an edelfosine receptor inHeLa cells can be activated to induce NF-κB/IL-8.

Functional assays were developed to identify receptors as NF-κB inducingreceptors, in particular, to identify lysolipid (LL) receptors, EDGreceptors and edelfosine receptors. In particular, assays were developedto measure NF-κB , IL-8 or IL-6 production.

With respect to the LL receptor(s) and edelfosine receptor(s), an assaywas developed to determine the response of HeLa cells to LL (includingS1P and LPA) and edelfosine, respectively, to induce NF-κB/IL-8activation/production.

As exemplified below, 293-EBNA cells were used to transfect EDGreceptors. The transfected 293 EBNA cells were then exposed to specificligands (namely, S1P, SPC and LPA) and NF-κB or IL-8 were measured as anindication of the inflammatory response. Accordingly, using thesefunctional assays, it has now been determined that LPA, S1P and/or SPCbind to EDG-2, EDG-3, EDG-4, EDG-5 and EDG-6 to induce NF-κB and/or IL-8(See FIG. 25). Since NF-κB and/or IL-8 are products of an inflammatoryresponse pathway and NF-κB is also associated with an anti-apoptoticpathway, EDG-2, EDG-3, EDG-4, EDG-5 and EDG-6 are receptors which arelinked to these same pathways. Thereby, by modulating these edgreceptors or any edg receptors which activate NF-κB, an inflammatoryresponse or apoptosis-modulating signal can be modulated.

The assays described herein are able to identify inflammatory EDG/LLreceptors both in heterologous expression and endogenous expressionsettings, and to aid in their cloning and characterization. Thus, EDG-2,EDG-3, EDG-4, EDG-5 and EDG-6 were identified herein as inflammatory LLreceptors through this approach. Similarly, the determination thatedelfosine can provoke a PTX-sensitive IL-8 response in HeLa cellssuggests that an edelfosine receptor resides in HeLa cells, which may ormay not correspond to an EDG or LL receptor. Isolation of this and otherEDG/LL receptors is a straightforward technical exercise, in light ofthe current disclosure. Given the demonstrated clinical effects ofedelfosine, a LL-derived anti-neoplastic agent, such isolated receptorsand the attendant functional assays offer great scientific, commercialand medical potential.

The non-receptor-dependent actions of LL might be expected to cause cellinjury, possibly activating NF-κB without a requirement for a GPCRreceptor. Therefore, a parallel assessment of cytotoxicity withfunctional response was conducted, along with a clear demonstration oftime- and concentration-dependence and ligand specificity, and anassessment of signal transduction mechanism, in order to validate NF-κBactivation as a functional assay for the receptors herein. (See Examplesbelow.)

The invention relates in another respect to polynucleotides, in theirisolated form, that encode the human EDG-4 receptor. The activity ofEDG-4 receptor can be measured using a variety of appropriate functionalassays, some of which are described hereinbelow. More particularly, theEDG-4 receptor is capable of binding with LLs, such as S1P and SPC, forsignal transduction to induce NF-κB and IL-8.

As used herein and designated by the upper case abbreviation, EDG,refers to the receptor in either naturally occurring or synthetic formand edg refers to the nucleotide sequence of the receptor. Inparticular, HEDG-4 refers to the human EDG-4 receptor homolog in eithernaturally occurring or synthetic form and hedg-4 refers to thenucleotide sequence of the human receptor. The HEDG-4 receptor isactivated by S1P and SPC and includes the amino acid sequence of FIG.16A or 16B and biologically active fragments thereof. More particularly,the HEDG-4 receptors preferably have at least 91% sequence identity witheach other, and more preferably at least 95% sequence identity with eachother.

Definitions

The following definitions are used herein for the purpose of describingparticular terms used in the application. Any terms not specificallydefined should be given the meaning commonly understood by one ofordinary skill in the art to which the invention pertains.

As used herein “isolated” means separated from nucleotide sequences thatencode other proteins. In the context of polynucleotide libraries, forinstance, a hedg-4 receptor-encoding nucleotide sequence is considered“isolated” when it has been selected, and hence removed from associationwith other nucleotide sequences within the library. Such nucleotidesequences may be in the form of RNA, or in the form of DNA includingcDNA, genomic DNA and synthetic DNA.

As used herein “purified” refers to sequences that are removed fromtheir natural environment, and are isolated or separated, and are atleast 60% free, preferably 75% free, and most preferably 90% free fromother components with which they are naturally associated.

An “oligonucleotide” is a stretch of nucleotide residues, which has asufficient number of bases to be used as an oligomer, amplimer or probein a polymerase chain reaction (PCR). Oligonucleotides are prepared fromgenomic or cDNA sequence and are used to amplify, reveal or confirm thepresence of a similar DNA or RNA in a particular cell or tissue.Oligonucleotides or oligomers comprise portions of a DNA sequence havingat least about 10 nucleotides and as many as about 35 nucleotides,preferably about 25 nucleotides.

“Probes” may be derived from naturally occurring, recombinant, orchemically synthesized single—or double—stranded nucleic acids or bechemically synthesized. They are useful in detecting the presence ofidentical or similar sequences.

A “portion” or “fragment” of a nucleotide or nucleic acid sequencecomprises all or any part of the sequence having fewer nucleotides thanabout 6 kb, preferably fewer than about 1 kb. A portion or fragment canbe used as a probe. Such probes may be labeled with reporter moleculesusing nick translation, Klenow fill-in reaction, PCR or other methodswell known in the art. To optimize reaction conditions and to eliminatefalse positives, nucleic acid probes may be used in Southern, Northernor in situ hybridizations to determine whether DNA or RNA encodingHEDG-4 is present in a cell type, tissue, or organ.

“Reporter” molecules are those radionuclides , enzymes, fluorescent,chemiluminescent, or chromogenic agents which associate with, establishthe presence of, and may allow quantification of a particular nucleotideor amino acid sequence.

“Recombinant nucieotide variants” encoding HEDG-4 may be synthesized bymaking use of the “redundancy” in the genetic code. Various codonsubstitutions, such as the silent changes which produce specificrestriction sites or codon usage-specific mutations, may be introducedto optimize cloning into a plasmid or viral vector or expression in aparticular prokaryotic or eukaryotic host system, respectively.

“Chimeric” molecules may be constructed by introducing all or part ofthe nucleotide sequence of this invention into a vector containingadditional nucleic acid sequence which might be expected to change anyone (or more than one) of the following HEDG-4 characteristics: cellularlocation, distribution, ligand-binding affinities, interchainaffinities, degradation/tumover rate, signaling, etc.

“Biologically Active or Active” refers to those forms, fragments, ordomains of any HEDG-4 polypeptide which retain at least some of thebiological and/or antigenic activities of any naturally occurringHEDG-4.

“Naturally occurring HEDG-4” refers to a polypeptide produced by cellswhich have not been genetically engineered and specifically contemplatesvarious polypeptides arising from polymorphisms found among humanpopulations, as well as those arising from RNA editing, alternativesplicing, or post-translational modifications of the polypeptideincluding but not limited to acetylation, carboxylation, glycosylation,plaosphorylation, lipidation and acylation.

“Derivative” refers to those amino acid sequences and nucleotidesequences which have been chemically modified. Such techniques forpolypeptide derivatives include: ubiquitination; labeling (see above);pegylation (derivatization with polyethylene glycol); and chemicalinsertion or substitution of amino acids such as ornithine which do notnormally occur in human proteins. A nucleotide sequence derivative wouldencode the amino acid which retains its essential biologicalcharacteristics of the natural molecule.

“Recombinant polypeptide variant” refers to any polypeptide whichdiffers from naturally occurring HEDG-4 by amino acid insertions,deletions and/or substitutions, created using recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added or deleted without abolishing activities of interest maybe found by comparing the sequence of HEDG-4 with that of relatedpolypeptides and minimizing the number of amino acid sequence changesmade in highly conserved regions.

Amino acid “substitutions” are conservative in nature when they resultfrom replacing one amino acid with another having similar structuraland/or chemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined byproducing the peptide synthetically or by systematically makinginsertions, deletions, or substitutions of nucleotides in the hedge-4sequence using recombinant DNA techniques.

A “signal or leader sequence” can be used, when desired, to direct thepolypeptide through a membrane of a cell. Such a sequence may benaturally present on the polypeptides of the present invention orprovided from heterologous sources by recombinant DNA techniques.

An “oligopeptide” is a short stretch of amino acid residues and may beexpressed from an oligonucleotide. It may be functionally equivalent toand the same length as (or considerably shorter than) a “fragment”,“portion”, or “segment” of a polypeptide. Such sequences comprise astretch of amino acid residues of at least about 5 amino acids and oftenabout 17 or more amino acids, typically at least about 9 to 13 aminoacids, and of sufficient length to display biological and/or antigenicactivity.

“Inhibitor” is any substance which retards or prevents a biochemical,cellular or physiological reaction or response. Common inhibitorsinclude but are not limited to antisense molecules, antibodies, andantagonists.

“Standard” is a quantitative or qualitative measurement for comparison.It is based on a statistically appropriate number of normal samples andis created to use as a basis of comparison when performing diagnosticassays, running clinical trials, or following patient treatmentprofiles.

“Stringent conditions” is used herein to mean conditions that allow forhybridization of substantially related nucleic acid sequences. Suchhybridization conditions are described by Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.Generally, stringency occurs within a range from about 5° C. below themelting temperature of the probe to about 20° C.-25° C. below themelting temperature. As understood by ordinary skilled persons in theart, the stringency conditions may be altered in order to identify ordetect identical or related nucleotide sequences. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.) and the concentration of the salts and othercomponents (e.g. the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency.

“Animal” as used herein may be defined to include human, domestic (catsdogs, etc.), agricultural (cows, horses, sheep, etc.) or test species(mouse, rat, rabbit, etc.).

“Nucleotide sequences” as used herein are oligonucleotides,polynucleotides, and fragments or portions thereof, and are DNA or RNAof genomic or synthetic origin which may be single or double stranded,and represent the sense or complement or antisense strands.

“Sequence Identity” is known in the art, and is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences, particularly, asdetermined by the match between strings of such sequences. Sequenceidentity can be readily calculated by known methods (ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity between two sequences, the term is wellknown to skilled artisans (see, for example, Sequence Analysis inMolecular Biology; Sequence Analysis Primer; and Carillo, H., andLipman, D., SIAM J. Applied Math., 48: 1073 (1988)). Methods commonlyemployed to determine identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988) or, preferably, in Needleman and Wunsch,J. Mol. Biol., 48: 443-445, 1970, wherein the parameters are as set inversion 2 of DNASIS (Hitachi Software Engineering Co., San Bruno,Calif.). Computer programs for determining identity are publiclyavailable. Preferred computer program methods to determine identitybetween two sequences include, but are not limited to, GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990)). The BLASTX program is publicly availablefrom NCBI (blast@ncbi.nlm.nih.gov) and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Bio. 215: 403-410 (1990)). Computational Molecular Biology,Lesk, A. M, ed. Unless specified otherwise in the claims, the percentidentity for the purpose of interpreting the claims shall be calculatedby the Needleman and Wucnsch algorithm with the parameters set inversion 2 of DNASIS.

The EDG receptor family of T7G receptors has been subdivided into 2subgroups on the basis of sequence similarity and genomic organization(Chun, Contos & Munroe, in press). We have determined that EDG-2, EDG-5(see U.S. Ser. No. 08/997,803, inorporated herein by reference) andEDG-6 (Genbank Accession AF011466) respond to LPA as an agonist, andshare a common intron structure within their coding regions. EDG-1,EDG-3, rat EDG-4/H218 (Accession U10699) and EDG-7 (see co-pending U.S.patent application Ser. No. 60/070,184) have intronless coding regionsand respond to S1P and SPC as agonists. The present T7G receptor,HEDG-4, has no intron within the coding region.

One aspect of the present invention is a method for using recombinantHEDG-4 receptors in an assay for screening ligands and potential drugcandidates. Although the use of T7G receptors in high-throughputscreening is well-known, no such screen has been reported for the HEDG-4receptor. More specifically, the novel HEDG-4 receptor presented hereincan be used to identify and rank the relative potency and efficacy ofpotential agonists. These compounds may be useful inasmuch as they wouldbe expected to modulate cellular or physiological responses to HEDG-4agonists, or to initiate or supplement HEDG-4 signaling in cells wherethe receptor occurs. Equally, once a quantitative and reliable assay isestablished, it can readily be applied to identify and rank the relativepotency and efficacy of receptor antagonists. This application, withoutlimiting other aspects, of the screening methods described herein isspecifically contemplated and incorporated within the scope of thisinvention.

It was determined that S1P and SPC are agonists for HEDG-4.

Other HEDG-4 ligands are likely to be found among the phospholipid classof compounds. Therefore, in one embodiment, phospholipid molecules couldbe screened to identify ligands. Particularly, it is believed thatpotential ligands include fatty acid chains of differing length, such as16, 17, 18, 19, 20, 22 and 24 carbon units, with or without 1, 2, 3 or 4unsaturated carbon-carbon bonds.

The nucleotide sequences encoding HEDG-4 (or their complement) havenumerous applications in techniques known to those skilled in the art ofmolecular biology. These techniques include use as hybridization probes,use in the construction of oligomers for PCR, use for chromosome andgene mapping, use in the recombinant production of HEDG-4, and use ingeneration of antisense DNA or RNA, their chemical analogs and the like.Uses of nucleotides encoding HEDG-4 disclosed herein are exemplary ofknown techniques and are not intended to limit their use in anytechnique known to a person of ordinary skill in the art. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown, e.g., the triplet genetic code, specific base pair interactions,etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of hedg-4 encodingnucleotide sequences may be produced. Some of these will only bearminimal homology to the nucleotide sequence of the known and naturallyoccurring hedg-4. The invention has specifically contemplated each andevery possible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurringhedg-4, and all such variations are to be considered as beingspecifically disclosed.

Although the nucleotide sequences which encode HEDG-4, its derivativesor its variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring hedg-4 under stringent conditions,it may be advantageous to produce nucleotide sequences encoding HEDG-4or its derivatives possessing a substantially different codon usage.Codons can be selected to increase the rate at which expression of thepeptide occurs in a particular prokaryotic or eukaryotic expression hostin accordance with the frequency with which particular codons areutilized by the host. Other reasons for substantially altering thenucleotide sequence encoding HEDG-4 and/or its derivatives withoutaltering the encoded amino acid sequence include the production of RNAtranscripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

Human genes often show considerable actual polymorphism; that is,variation in nucleotide sequence among a fraction of the entire humanpopulation. In many cases this polymorphism can result in one or moreamino acid substitutions. While some of these substitutions show nodemonstrable change in function of the protein, others may producevarying degrees of functional effects. In fact, many natural orartificially produced mutations can lead to expressible HEDG proteins.Each of these variants, whether naturally or artificially produced, isconsidered to be equivalent and specifically incorporated into thepresent invention.

Nucleotide sequences encoding HEDG-4 may be joined to a variety of othernucleotide sequences by means of well established recombinant DNAtechniques (Sambrook J et al (1989) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.; orAusubel F M et al (1989) Current Protocols in Molecular Biology, JohnWiley & Sons, New York City). Useful nucleotide sequences for joining tohedg-4 include an assortment of cloning vectors such as plasmids,cosmids, lambda phage derivatives, phagemids, and the like. Vectors ofinterest include expression vectors, replication vectors, probegeneration vectors, sequencing vectors, etc. In general, vectors ofinterest may contain an origin of replication functional in at least oneorganism, convenient restriction endonuclease senstive sites, andselectable markers for one or more host cell systems.

Another aspect of the subject invention is to provide for hedg-4specific hybridization probes capable of hybridizing with naturallyoccurring nucleotide sequences encoding HEDG-4. Such probes may also beused for the detection of similar T7G encoding sequences and shouldpreferably contain at least 91% nucleotide identity to hedg-4 sequenceand more preferably at least 95% identity. The hybridization probes ofthe subject invention may be derived from the nucleotide sequencepresented in the figures for hedge or from genomic sequences includingpromoter, enhancers, introns or 3′-untranslated regions of the nativegene. Hybridization probes may be labeled by a variety of reportermolecules using techniques well known in the art. Preferably, thehybridization probes incorporate at least 15 nucleotides, and preferablyat least 25 nucleotides, of the hedg-4 receptor.

It will be recognized that many deletional or mutational analogs ofnucleic acid sequences for HEDG-4 will be effective hybridization probesfor HEDG-4 nucleic acid. Accordingly, the invention relates to nucleicacid sequences that hybridize with such HEDG-4 encoding nucleic acidsequences under stringent conditions.

Stringent conditions will generally allow hybridization of sequence withat least about 70% sequence identity, more preferably at least about80-85% sequence identity, even more preferably at least about 90%sequence identity, and most preferably with at least about 95% sequenceidentity Hybridization conditions and probes can be adjusted inwell-characterized ways to achieve selective hybridization ofhuman-derived probes. Nucleic acid molecules that will hybridize toHEDG-4 encoding nucleic acid under stringent conditions can beidentified functionally, using methods outlined above, or by using forexample the hybridization rules reviewed in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.Without limitation, examples of the uses for hybridization probesinclude: histochemical uses such as identifying tissues that expressHEDG-4; measuring mRNA levels, for instance to identify a sample'stissue type or to identify cells that express abnormal levels of HEDG-4;and detecting polymorphisms in the HEDG-4. RNA hybridization proceduresare described in Maniatis et al. Molecular Cloning, a Laboratory Manual(Cold Spring Harbor Press, 1989). PCR as described U.S. Pat. Nos.4,683,195; 4,800,195; and 4,965,188 provides additional uses foroligonucleotides based upon the nucleotide sequence which encodes theEDG-4 sequences of the invention. Such probes used in PCR may be ofrecombinant origin, chemically synthesized, or a mixture of both.Oligomers may comprise discrete nucleotide sequences employed underoptimized conditions for identification of hedg-4 in specific tissues ordiagnostic use. The same two oligomers, a nested set of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for identification of closely related DNA's or RNA's. Rulesfor designing PCR primers are now established, as reviewed by PCRProtocols, Cold Spring Harbor Press, 1991. Degenerate primers, i.e.,preparations of primers that are heterogeneous at given sequencelocations, can be designed to amplify nucleic acid sequences that arehighly homologous to, but not identical to hedg-4. Strategies are nowavailable that allow for only one of the primers to be required tospecifically hybridize with a known sequence. See, Froman et al., Proc.Natl. Acad. Sci. USA 85: 8998, 1988 and Loh et al., Science 243: 217,1989. For example, appropriate nucleic acid primers can be ligated tothe nucleic acid sought to be amplified to provide the hybridizationpartner for one of the primers. In this way, only one of the primersneed be based on the sequence of the nucleic acid sought to beamplified. PCR methods of amplifying nucleic acid will utilize at leasttwo primers. One of these primers will be capable of hybridizing to afirst strand of the nucleic acid to be amplified and of primingenzyme-driven nucleic acid synthesis in a first direction. The otherwill be capable of hybridizing the reciprocal sequence of the firststrand (if the sequence to be amplified is single stranded, thissequence will initially be hypothetical, but will be synthesized in thefirst amplification cycle) and of priming nucleic acid synthesis fromthat strand in the direction opposite the first direction and towardsthe site of hybridization for the first primer. Conditions forconducting such amplifications, particularly under preferred stringenthybridization conditions, are well known. See, for example, PCRProtocols, Cold Spring Harbor Press, 1991.

Other means of producing specific hybridization probes for hedg-4include the cloning of nucleic acid sequences encoding HEDG-4 or HEDG-4derivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate reporter molecules.

It is possible to produce a DNA sequence, or portions thereof, entirelyby synthetic chemistry. After synthesis, the nucleic acid sequence canbe inserted into any of the man) available DNA vectors and theirrespective host cells using techniques which are well known in the art.Moreover, synthetic chemistry may be used to introduce mutations intothe nucleotide sequence. Alternately, a portion of sequence in which amutation is desired can be synthesized and recombined with longerportion of an existing genomic or recombinant sequence.

The nucleotide sequence for hedg-4 can be used in an assay to detectinflammation or disease associated with abnormal levels of HEDG-4expression. The cDNA can be labeled by methods known in the art, addedto a fluid, cell or tissue sample from a patient, and incubated underhybridizing conditions. After an incubation period, the sample is washedwith a compatible fluid which optionally contains a reporter molecule.After the compatible fluid is rinsed off, the reporter molecule isquantitated and compared with a standard as previously defined.

A diagnostic test for aberrant expression of HEDG-4 can acceleratediagnosis and proper treatment of abnormal conditions of for example,the heart, kidney, lung and testis. Specific examples of conditions inwhich aberrant expression of HEDG-4 may play a role include adultrespiratory distress, asthma, rheumatoid arthritis, cardiac ischemia,acute pancreatitis, septic shock, psoriasis, acute cyclosporinenephrotoxicity and early diabetic glomerulopathy, as well as lung damagefollowing exposure to cigarette smoke, asbestos or silica.

Nucleotide sequences encoding hedg-4 may be used to produce a purifiedoligo—or polypeptide using well known methods of recombinant DNAtechnology. Goeddel (1990, Gene Expression Technology, Methods andEnzymology, Vol. 185, Academic Press, San Diego Calif.) is one amongmany publications which teach expression of an isolated nucleotidesequence. The oligopeptide may be expressed in a variety of host cells,either prokaryotic or eukaryotic. Host cells may be from the samespecies from which the nucleotide sequence was derived or from adifferent species. Advantages of producing an oligonucleotide byrecombinant DNA technology include obtaining adequate amounts of theprotein for purification and the availability of simplified purificationprocedures.

Cells transformed with DNA encoding HEDG-4 may be cultured underconditions suitable for the expression of T7Gs, their extracellular,transmembrane or intracellular domains and recovery of such peptidesfrom cell culture. HEDG-4 (or any of its domains) produced by arecombinant cell may be secreted, expressed on cellular membranes or maybe contained intracellularly, depending on the particular geneticconstruction used. In general, it is more convenient to preparerecombinant proteins in secreted form. Purification steps vary with theproduction process and the particular protein produced. Often anoligopeptide can be produced from a chimeric nucleotide sequence. Thisis accomplished by ligating the nucleotides from hedge or a desiredportion of the polypeptide to a nucleic acid sequence encoding apolypeptide domain which will facilitate protein purification (Kroll D Jet al (1993) DNA Cell Biol. 12:441-53).

In addition to recombinant production, fragments of HEDG-4 may beproduced by direct peptide synthesis using solid-phase techniques (e.g.Stewart at al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., SanFrancisco QA; Merrifield J (1963) J Am Chem. Soc. 85:2149-2154).Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordancewith the instructions provided by the manufacturer. Additionally, aparticular portion of HEDG-4 may be mutated during direct synthesis andcombined with other parts of the peptide using chemical methods.

HEDG-4 for antibody induction does not require biological activity:however, the protein must be antigenic. Peptides used to induce specificantibodies may have an aa (amino acid) sequence consisting of at leastfive amino acids (aa), preferably at least 10 aa. They should mimic aportion of the aa sequence of the protein and may contain the entire aasequence of a small naturally occurring molecule such as HEDG-4. Anantigenic portion of HEDG-4 may be fused to another protein such askeyhole limpet hemocyanin, and the chimeric molecule used for antibodyproduction.

Antibodies specific for HEDG-4 may be produced by inoculation of anappropriate animal with the polypeptide or an antigenic fragment. Anantibody is specific for HEDG-4 if it is produced against an epitope ofthe polypeptide and binds to at least part of the natural or recombinantprotein. Antibody production includes not only the stimulation of animmune response by injection into animals, but also analogous processessuch as the production of synthetic antibodies, the screening ofrecombinant immunoglobulin libraries for specific-binding molecules(e.g. Orlandi R et al (1989) PNAS 86:3833-3837, or Huse WD et al (1989)Science 256:1275-1281) or the in vitro stimulation of lymphocytepopulations. Current technology (Winter G and Mistein C (1991) Nature349:293-299) provides for a number of highly specific binding reagentsbased on the principles of antibody formation. These techniques may beadapted to produce molecules which specifically bind HEDG-4s.

An additional embodiment of the subject invention is the use of HEDG-4specific antibodies, inhibitors, ligands or their analogs as bioactiveagents to treat inflammation or disease including, but not limited toviral, bacterial or fungal infections; allergic responses; mechanicalinjury associated with trauma; hereditary diseases; lymphoma orcarcinoma; or other conditions which activate the genes of kidney, lung,heart, lymphoid or tissues of the nervous system.

Bioactive compositions comprising agonists, antagonists, receptors orinhibitors of HEDG-4 may be administered in a suitable therapeutic dosedetermined by any of several methodologies including clinical studies onmammalian species to determine maximal tolerable dose and on normalhuman subjects to determine safe dose. Additionally, the bioactive agentmay be complexed with a variety of well established compounds orcompositions which enhance stability or pharmacological properties suchas half-life. It is contemplated that the therapeutic, bioactivecomposition may be delivered by intravenous infusion into thebloodstream or any other effective means which could be used fortreating problems involving aberrant expression of the EDG-4 gene.

All publications and patent applications mentioned herein areincorporated by reference for the purpose of describing themethodologies, cell lines and vectors, among other things. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure, for example, by virtue ofprior invention.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLE 1 IL-8 Response to S1P in HeLa Cells is Concentration and TimeDependent

A preliminary survey of cell lines for IL-8 and IL-6 response to S1Pidentified HeLa cells as a potential responder (FIG. 1B), while HL-60cells were unresponsive, consistent with the reported lack of S1Preceptors in these cells (FIG. 5). IL-8 and IL-6 are potently induced bya variety of proinflammatory agents, including TNF-α, phorbol ester(TPA) and ultraviolet radiation. Induction by these agents is dependenton transcriptional upregulation by NF-κB, although NF-IL6 and AP-1 alsoplay roles in certain experimental models. Because commerciallyavailable IL-8 ELISA kits offer a robust and simple measurement withmoderately high throughput, we chose to focus on the IL-8 response inthe first instance. Later work included the NF-κB reporter gene.However, since the novelty and utility of this invention broadlyencompasses inflammatory signaling by edg/LL receptors, we include otherreceptor-dependent proinflammatory reporters, including, but not limitedto NF-κB, NF-IL6 and AP-1 activation are within the scope of the presentinvention.

Procedure #1 For HeLa Cells

A. Seeding Cells and Cell Plating Density

Cells: HeLa (adenocarcinoma, human)

Media: DMEM/F12+10%FBS adherent

1) Cells were seeded at 0.2×10⁶ cells/well in 6-well plates.

2) Confluency of cells after 24-32 hrs was between 60-70%.

B. Overnight Serum-Starvation

1) Media was aspirated (no PBS wash).

2) 1.5 ml 0.5% FBS media was added to each well.

C. Treatments and Collection

1) Made up all required solutions in 0.5% FBS media (control). Handlingof LL for use in NF-κB experiments requires that sonication, commonlyused to resuspend LPA, not be done; NF-κB may be activated by lipidperoxides created through vigorous frothing.

Solutions:

TPA 100 ng/ml, Stock 0.1 mg/ml in DMSO Sigma, Cat. P-1585, Dilution1:1000

LPA 10 μM, Stock 10 mM in 0.2% Albumin Bovine, Sigma, Cat. A-0281in PBS;Dilution 1:1000

LPA 1 μM, Dilute 10 μM 1:10

S1P 10 μM, Stock 10 mM in methanol, Sigma, Cat. S-9666, Dilution 1:1000

S1P 1 μM, Dilute 10 μM 1:10

Note: All stock solutions are dissolved by pipetting and stored at −20°C.

2) Media was aspirated.

3) 1.5 ml appropriate treatments were added to each well.

4) All plates were placed at 37° C./5% CO₂ for either 1, 6, or 24 hours.

5) After the specified time cell supernatants were collected into 1.5 mleppendorf tubes, spun down at 14000 rpm for 5 minutes and stored at −20°C. for later ELISA determination.

D. Detection of Interleukin-8 (IL-8) using an IL-8 ELISA (Enzyme-LinkedInimunoSorbent Assay).

1) The Quantikine Human IL-8 ImmunoAssay Kit was obtained from R&DSystems (Cat. D8050).

2) The kit and all samples were allowed to equilibrate to roomtemperature prior to use.

3) All reagents were provided in the kit and prepared according to theinstructions provided.

4) The assay procedure was followed as recommended in the kit for cellculture supernatant samples.

5) ELISA was performed on 50 μl samples of culture supernatant andduplicate samples were measured for each well. Each treatment wasperformed on triplicate wells.

6) Plates were read on UVmax kinetic microplate reader (MolecularDevices), set to 450 nm and correction set to 575 nm, using Wsoftmaxsoftware version 2.34.

Results: This experiment showed a time- and concentration-dependent IL-8response to S1P, but not LPA, in HeLa cells (see FIG. 1B).

EXAMPLE 2 S1P and SPC Both Induce a Concentration-dependent,PTX-sensitive IL-8 Response in HeLa Cells

S1P and SPC both show PTX-sensitive functional responses in certain celltypes. However, in some cell types S1P shows 10-fold or higher potencythan SPC, while in other cell types S1P and SPC are roughly equipotent.If the IL-8 response to S1P and SPC is receptor-mediated, we mightexpect to see PTX-sensitivity with both ligands and possibly, an equalor reduced potency with SPC.

Procedure #2 For HeLa Cells

A. Seeding Cells and Cell Plating Density

Cells: HeLa (adenocarcinoma, human), Media: DMEM/F12+10%FBS adherentcells

1) Cells were seeded at 2.5×10⁴ cells/well in 24-well plates.

2) Confluency of cells after 24-32 hrs was between 60-70%.

B. Overnight Serum-Starvation and PTX Pre-Treatment

1) Media was aspirated (no PBS wash).

2) 0.5 ml 0.5% FBS media was added to all wells not requiring PTXpre-treatment.

3) For wells requiring PTX; 0.5 ml 0.5% FBS media containing 50 ng/mlPTX (1 volume PTX (RBI Cat. P140): 1 volume DTT, incubate 37° C. for 30minutes then dilute to 50 ng/ml) was added.

C. Treatments and Collection

1) Made up all required solutions in 0.5% FBS media (control).

Solutions:

S1P 3, 10, 30, 100, 300, 1000, 3000, 10000 nM

SPC 10 μM Stock 10 mM in methanol, Sigma, Cat. S4257, Dilution 1:1000

SPC 1, 3, 10, 30, 100, 300, 1000, and 3000 nM

2) Media was aspirated.

3) 0.5 ml appropriate treatments were added.

4) All plates were placed at 37° C./5% CO₂ for 6 hours.

5) After the specified time cell supematants were collected into 1.5 mleppendorf tubes, spun down at 14000 rpm for 5 minutes and stored at −20°C. for later ELISA determination.

D. Refer to Procedure #1 For HeLa Cells (D).

Results: The experiment demonstrated unequivocally that both S1P and SPCcan induce IL-8 in HeLa cells in a concentration-dependent manner (FIG.2A), and that these responses are PTX-sensitive, as expected of aG_(i)-coupled receptor (see FIG. 2B).

EXAMPLE 3 Effect of PTX on IL-8 Response to S1P and TNF-α in HeLa Cells

Effects of PTX toxin reflect a requirement for the G_(i/o) family ofheterotrimeric G proteins, which play critical roles in the multipleactions of GPCRs. It is possible, however, that the PTX inhibition ofS1P-induced IL-8 response reflects an indirect effect on downstreamsignal transduction events, rather than an effect on the G proteinsdirectly coupled to a GPCR for S1P. If a general block of IL-8production is produced by PTX in HeLa cells, then IL-8 production byTNF-α should also be inhibited. TNF-α induces IL-8 through its ownreceptor, which is not a GPCR and does not require G_(i/o) forsignaling. On the other hand, if the IL-8 response to TNF-α isunaffected, then the blockade by PTX is specific to S1P but not TNF-αsignaling pathways.

Procedure #3 For HeLa Cells

Follow Procedure #2 For HeLa Cells with the following exceptions:

1) Solutions required in section C are as follows:

S1P 5 μM

TNF-α 50 ng/ml Stock 10 μg/ml in 0.1% Albumin Bovine R&D, Cat. 210-TA

(Albumin: Sigma; Cat. A-028 1) in PBS

Dilute 1:200

Results: The results clearly showed that while PTX potently blocked theIL-8 response to S1P, the response to TNF-α was not significantlyaffected (see FIG. 3). Thus, G_(i/o) pathways are required for S1Psignaling that leads to the IL-8 response in HeLa cells.

EXAMPLE 4A IL-8 Response to S1P in HeLa Cells is Ligand-selective andnot a General LL Response

S1P shares a detergent-like structure with many other LL. (See FIG. 1A)Thus, non-specific activation of NF-κB by cell injury or membraneactions of S1P should be produced by many other LL as well.Additionally, any general non-selective LL receptor expressed in HeLashould be activated interchangeably by several different LL.Alternatively, ligand-selective activation of NF-κB argues for areceptor-mediated mechanism amenable to future drug discovery.

Procedure #4 For HeLa Cells

Follow Procedure #2 For HeLa Cells with the following exceptions:

1) No PTX Pre-Treatment is required in section B.

2) Solutions required in section C are as follows:

LPC Stock 10 mM in methanol Sigma, Cat. L-1381

LPE Stock 10 mM in chloroform Sigma, Cat. L-4754

LPG Stock 10 mM in methanol Sigma, Cat. L-4525

LPI Stock 10 mM in 1% Albumin Bovine in PBS Sigma, Cat. L-7635

LPS Stock 10 mM in 0.2% Albumin Bovine in PBS Sigma, Cat. L-5772

Lyso-PAF Stock 10 mM in 1% Albumin Bovine in PBS Sigma, Cat. L-7890

Lysosulfatide Stock 10 mM in DMSO Sigma, Cat. L-3640

Sphingosine Stock 10 mM in methanol Sigma, Cat. S-6136

Sphingomyelin (SM) Stock 10 mM in methanol Sigma, Cat. S-7004

Concentrations for LPC, LPE, LPG, LPS, sphingosine and SM used were 10,50, 100, 1000, and 5000 nM. Concentrations for LPI, lyso-PAF andlysosulfatide used were 0.3 and 3 μM.

Results: Only S1P and SPC significantly induced IL-8 production,strongly suggesting that a ligand-selective receptor mediates thePTX-sensitive IL-8 response pathway. While sphingosine is shown togetherwith S1P as examples of the ligand-selectivity of the IL-8 response, asimilar lack of response was observed in HeLa cells with all othercompounds listed above, but not shown on the graph (see FIG. 4A).

EXAMPLE 4B IL-8 Response to S1P, LPA and Other Lysolipids in PrimaryCultured Human Umbilical Vein Endothelial Cells (HUVEC)

While HeLa cells form the basis of an experimentally homogeneous assaysystem, these cells have been carried continuously in culture for manyyears. Moreover, they are a transformed (i.e. neoplastic) cell line, andas such, carry many chromosomal and genetic abnormalities. As will bereadily apparent to one skilled in cell and molecular biology, findingsin HeLa cells should be confirmed in a non-transformed cell line,preferably primary cultured human cells. We chose HUVEC, a commonlyavailable human primary cell culture. Since these cells are derived fromthe endothelium lining the umbilical vein, they share manycharacteristics and response pathways with endothelial cells foundelsewhere in the human body. More particularly, HUVEC cells have beenused for the study of NF-κB activation by GPCRs (Ishizuka T, et alStimulation with thromboxane A2 (TXA2) receptor agonist enhances ICAM-1,VCAM-1 or ELAM-1 expression by human vascular endothelial cells. ClinExp Immunol. 1998 June;112(3):464-470; Munoz C, et al Pyrrolidinedithiocarbamate inhibits the production of interleukin-6, interleukin-8,and granulocyte-macrophage colony-stimulating factor by humanendothelial in response to inflammatory mediators: modulation of NF-κBand AP-1 transcription factors activity. Blood. 1996 November1;88(9):3482-3490.). Among the documented consequences of NF-κBactivation in this cell type are the production of cytokines such asIL-8, IL-6 and GM-CSF. In addition, cell adhesion molecules such asVCAM-1, ELAM-1 and ICAM-1 are upregulated, which play distinct roles inthe attaclunent and extravasation of peripheral blood leukocytes atsites of injury or inflammation. The following experiment was conductedto look for IL-8 production in cultured HUVEC exposed to S1P, LPA orother lysolipids.

Plating, Pretreatment and Treatment of HUVEC

Procedures were followed as detailed above in “Procedure #1 for HeLaCells” with the following exceptions:

Cells: HUVEC (Clonetics, Cat. CC-2519) were passaged according tosupplier's instructions and used at passage 3. Cells were plated at20,000 cells/well into 24-well plates. The next day, cells wereserum-starved overnight in EBM medium (Clonetics) with 0.5% FBS, andthen treated in EBM without FBS for 6 hr with the following lysolipids:

1) Control (no lysolipids)

2) Anandarnide

3) Edelfosine

4) LPA

5) S1P

6) SPC

7) Psychosine

Supematants were collected and IL-8 levels were deternined using ELISAas described previously.

Results: After 6 hr of treatment with 5 μM S1P, IL-8 levels wereincreased approximately 5-fold over untreated controls, as shown in FIG.4B. LPA induced a 3-fold IL-8 increase at this concentration. Marginalincreases were seen after SPC and psychosine treatment, while noresponse was seen with anandamide or edelfosine. Therefore, IL-8production was responsive to S1P in primary cultured human endothelialcells, similar to the results seen in HeLa cells. In addition, LPAinduced IL-8 production in HUVEC, but not HeLa cells, suggesting thatinflammatory receptors for LPA may be expressed in the former cell type.As shown below in FIG. 23, three cloned edg receptors respond to LPA asan agonist, and all three appear to transduce NF-κB activation in anagonist-dependent manner.

EXAMPLE 5 Lack of IL-8 Response to S1P in HL-60 Cells

HL-60 cells have been reported not to possess S1P receptors. Onecontradictory report has been published, but in that work, 10 μMconcentration of S1P was used, 10-1000 times higher than other studiesof S1P receptors. Nonetheless, HL-60 cells were examined for IL-8response to S1P. As a control, IL-8 release from HL-60 cells was testedafter treatment with TNF-α, which acts through a non-GPCR cell-surfacereceptor.

Procedure for HL-60 Cells:

A. Seeding Cells and Cell Plating Density

Cells: HL-60 (promyelocytic, human) suspension cells

Media: RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 4.5g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate+10% FBS

1) Cells were plated at a density of 0.25×10⁶ cells/ml.

2) Density of cells after 48-56 hrs was approximately 1×10⁶ cells/ml.

B. Overnight Pre-Treatments

1) Cells were spun down at 1000 rpm for 5 minutes.

2) Cell pellcts were resuspended in 0.5% FBS media at a density ofapproximately 1×10⁶ cells/ml.

C. Treatments and Collection

1) Made up all required solutions in 0.5% FBS media (control).

TNF-α 10 ng/ml

LPA 10 and 1 μM

S1P 10 and 1 μM

2) 1.4 ml appropriate treatments were added to each well of a 6-wellplate.

3) Cells were spun down at 1000 rpm for 5 minutes.

4) Cells were resuspended in 0.5% media to give a density ofapproximately 1×10⁶ cells/100 μl.

5) 100 μl cell suspension was added to each well.

6) All plates were placed at 37° C./5% CO₂ for either 1, 6, or 24 hours.

7) After the specified time cell supematants were collected into 1.5 mleppendorf tubes, spun down at 14000 rpm for 5 minutes and stored at −20°C.

D. Refer to Procedure #1 For HeLa Cells (D).

Results: Although HL-60 cells were capable of responding at 6 or 24 hrto TNF-α by releasing IL-8, no such release occurred in response to S1Por LPA at concentrations up to 3 μM (see FIG. 5). This concentration is100 times higher than the lowest concentration that reliably inducesIL-8 production in HeLa cells. Thus, the IL-8 response to S1P isexpressed in some, but not all cell types.

EXAMPLE 6 HeLa Cell IL-8 Response to S1P is not Due to Cytotoxicity

For LL, demonstration of signaling at concentrations well below thosethat cause lo cytotoxicity is important. For this purpose, an experimentwas conducted to measure cytotoxicity in parallel with IL-8 response. Astringent measure of cytotoxicity was applied, in that IL-8 responseswere measured after 6 hr of S1P treatment, whereupon the medium wasreplaced with normal medium and viable cells were counted at 24 hr.Therefore, IL-8 production had to be robust to be observed at 6 hr,while even slight or delayed toxicity would be seen as a loss ofviability at 24 hr.

Procedure #5 For HeLa Cells

Follow Procedure #2 For HeLa Cells with the following exceptions:

1) No PTX Pre-Treatment is required in section B.

2) Solutions required in section C are as follows:

S1P 0.3, 1, 3, 10, and 30 μM.

3) Cytotoxicity determination was added to section C; after step 5, 0.5ml of 0.5% FBS/media was added to all the wells and placed at 37°C./5%CO₂ overnight.

4) Number of viable cells were counted after 24 hours of the initialtreatments.

Results: No loss of HeLa viability was seen 24 hr after treatment withS1P concentrations up to 10 μM. In contrast, IL-8 production was seeneven at 0.3 μM S1P, where levels were already near plateau values (seeFIG. 6). In repeated experiments, the lowest S1P concentration thatreliably induces IL-8 is about 30 nM, more than 100-fold below thecytotoxic threshold. HL-60 cells, on the other hand, show toxicitybeginning at 10 μM S1P, but fail to produce IL-8 below the cytotoxicthreshold. Thus, the IL-8 response to S1P does not reflect anon-specific cellular response to injury or impending death.

EXAMPLE 7 Effect of Suramin on IL-8 Response to S1P in HeLa Cells

Suramin is a non-selective inhibitor of extracellular ligand-receptorinteractions with no known intracellular targets. This agent is used toprovide evidence of an extracellular site of action both for LPA andS1P. The IL-8 response was tested to determine if it could be blocked atthis extracellular site.

Procedure #6 For HeLa Cells

Follow Procedure #2 For HeLa Cells with the following exceptions:

1) No PTX Pre-Treatment is required in section B.

2) Solutions required in section C are as follows:

3) Suramin 1 mg/ml, Stock 100 mg/ml in distilled water, Calbiochem, Cat.574625, Dilute

1:100

S1P 1 μM

S1P 1 μM+suramin 1 mg/ml

4) A 30 minute pre-treatment at 37° C./5%CO₂ of 0.5 ml of 1 mg/mlsuramin was done to all wells except control and S1P 1 μM before step 3of section C.

Results: Suramin was extremely effective in blunting the IL-8 responseto S1P (see FIG. 7). Therefore, the most likely site of S1P action is atan extracellular receptor.

EXAMPLE 8 Effect of NDGA and NAC on IL-8 Response to S1P in HeLa Cells

NF-κB and IL-8 production can be induced by many different inflammatoryagents. Nearly all these diverse agents initiate signal transductionpathways that ultimately converge on destruction of the intracellularrepressor IκB, which holds NF-κB function in check in resting cells.However, the upstream pathways used to target IκB differ depending onthe nature of the inducer. While inflammatory cytokines and TPA useintracellular reactive oxygen species (ROS) as a second messenger, TNF-αand IL-1 usually do not. The ROS pathway and subsequent NF-κB activationcan be inhibited by NDGA, NAC and certain other antioxidants. Therefore,the sensitivity of the IL-8 response induced by S1P to theseantioxidants was evaluated.

Procedure #7 For HeLa Cells

Follow Procedure #2 For HeLa Cells with the following exceptions:

1) No PTX Pre-Treatment is required in section B.

2) Solutions required in section C are as follows:

3) NDGA 40 μM Stock 10 mM in ethanol, Sigma, Cat. N-5023, Dilute 1:250

NAC 30 mM Stock 0.3 M in PBS, pH to 7.4, Calbiochem, Cat.106425, Dilute1:10

S1P 1 μm

S1P 1 μM+NDGA 40 μM

S1P 1 μM+NDGA 10 μM

S1P 1 μM+NAC 30 mM

4) A 30 minute pre-treatment at 37° C./5%CO₂ of 0.5 ml of either NDGA orNAC was done to all wells except control and S1P 1 μM before step 3 ofsection C.

Results: The IL-8 response to S1P was significantly inhibited by bothantioxidants (see FIG. 8). As noted in the literature, the lipophilicantioxidant NDGA, was more potent than the hydrophilic NAC. However,some toxicity of NDGA was seen at 40 μM, a concentration that completelyinhibited the IL-8 response to S1P. Nevertheless, these structurallyunrelated antioxidants both inhibited the IL-8 response to S1P,suggesting a cytokine-like pathway mediates S1P signal transduction.

EXAMPLE 9 Suramin and PTX-sensitive IL-8 Response to Edelfosine, anAlkyl Ether Lysophospholipid, in HeLa Cells

Edelfosine is an alkyl ether lysophospholipid with potent and selectiveantitumor activity. In spite of numerous studies highlighting changes ingene expression and signal transduction provoked by edelfosine,conflicting data have been reported on its mechanism of action.Edelfosine inhibits protein kinase C, and thus may have intracellularsites of action. Edelfosine also can inhibit NF-κB in at least some celltypes. Most important, edelfosine spares normal bone marrow cells atconcentrations which kill tumor cells. The mechanism by which thisdiscrimination is effected is unclear. However, given the structuralsimilarity to LPA, the possibility that edelfosine might act on an edgfamily or LL receptor was considered. Therefore an IL-8 response toedelfosine in HeLa cells in the presence or absence of PTX or suraminwas tested.

Procedure #8 For HeLa Cells

Follow Procedure #2 For HeLa Cells with the following exceptions:

1) Solutions required in section C are as follows:

Suramin 1 mg/ml

ET-18-OCH₃ 10 μM Stock 10 mM in ethanol Calbiochem, Cat. 341207 Dilute1:1000

ET-18-OCH₃ 1 μM Dilute 1:10

ET-18-OCH₃ 3 μM

ET-18-OCh₃ 3 μM+suramin 1 mg/ml

2) A 30 minute pre-treatment at 37° C./5%CO₂ of 0.5 ml of suramin wasdone to all wells except control, any PTX and ET-18-OCH₃ wells beforestep 3 of section C.

Results: Edelfosine, like S1P, induced an IL-8 response in HeLa cells atnon-cytotoxic concentrations (see FIG. 9). Moreover, this response waspotently inhibited by PTX and suramin, suggesting that a G_(i/o)-coupledcell-surface receptor may mediate the induction of IL-8 by edelfosine.This receptor may be an edg or LL GPCR, although interaction with apreviously identified PAF receptor cannot yet be ruled out. This findingcontradicted edelfosine's inhibition of NF-κB previously reported in adifferent cell type. The present invention offers the means to identifyand characterize the HeLa cell receptor for edelfosine. Expression ofthis receptor can then be compared in cells which differ in theircytotoxicity and NF-κB responses to edelfosine.

EXAMPLE 10A Heterologous Expression of EDG-4/H218 in COS-1 CellsReconstitutes the IL-8 Response to S1P

We used a cAMP inhibition assay to show the presence of functional S1Preceptors in Swiss 3T3, mouse neuronal B-103 and hamster CHO ProS cells.By comparing the cAMP responses of these cells to the expression profileof the 7 identified edg receptors, we speculated that both EDG-3 andEDGY are likely to be S1P receptors. However, although COS and HEK-293cells both express abundant RNA for EDG-3, neither cell line shows anIL-8 response to S1P. This suggested that EDGY might selectively mediatethe IL-8 response to S1P. Unfortunately, EDG-4 previously could not bemeasured in HeLa, COS-1 or other primate cells, since it has not yetbeen cloned from these species. The present invention remedies thissituation by providing the sequence of the cloned HEDG-4. However, bytransient transfection with a eukaryotic expression vector expressingfull-length rat edg-4 cDNA it could be determined if this edg receptorcan reconstitute the IL-8 response to S1P in COS-1 cells. The experimentincluded NF-κB reporter DNA to test for induction of the CAT reportergene in parallel with the IL-8 response.

A. DEAE/Dextran Cell Suspension Transient Transfection.

Transfection was done as described in Anal Biochem 218:460 (1994).

a) Solutions:

RSC: 49 ml RPMI 1640 (Gibco; Cat. 21870-076)+1 ml Fetal calf serum +50μl of 100 mM chloroquine (Sigma; Cat. C6628)

DEAE/RSC: 18.4 ml RSC+1.6 ml of 10 mg/ml DEAE/Dextran (Promega; Cat.E112A)

b) Transfection procedure:

1) 6 ml RSC was added to 4-50 ml tubes. The following amounts of DNAwere added:

DNA (μg)/tube Tube 1 2 3 4 pcDNA3 5 5 — — pC3-redg4 (rat edg-4) — — 5 56xNFκB-tk-CAT5 5 2 5 2 pBluescript — 3 — 3

The tubes were incubated at 37° C. until DEAE/RSC solution was made.

2) 6 ml of DEAE/RSC solution was added to each tube and incubated at 37°C. for 2 min.

3) 1.5 ml COS-1 cell suspension (5.5×10⁶ cells total) in RSC was addedto each tube and incubated for 105 min in 37° C. incubator. Tubes weremixed every 20 min.

4) Following incubation, tubes were spun for 5 min, cell pellets werewashed with DMEM/F12+10% FBS once and then resuspended in 10 ml media.Cells were plated in 24-well plates at 0.2×10⁶ cells/well.

B. Treatment.

After 2 days (˜40 hrs), cells were serum-starved (0.5% FBS media) withor without PTX (50 ng/ml) for at least 6 hrs and treated overnight with0.5% FBS media, S1P (5 μm) in 0.5% FBS media or TPA (100 ng/ml) in 0.5%FBS media. 500 μl treatment volume was used. Supernatants weremicrofuged at 14,000 rpm for 10 min., transferred to new eppendorf tubesand stored at −20° C. for future IL-8 ELISA determination.

C. IL-8 ELISA (Enzyme-Linked ImmunoSorbent Assay).

The procedure as outlined in Procedure for HeLa Cells (D) was followed,using 50 μl of sample per ELISA determination in duplicate.

Results: COS-1 cells transfected with the EDG-4 expression plasmidshowed a 2-fold increase in IL-8 release when treated with 5 μm S1P ascompared to untreated cells (see FIG. 1A.). No IL-8 response to Sip wasseen in control cells transfected with the empty expression vectorpcDNA3. Moreover, the IL-8 response to S1P in EDG-4 transfected cellswas pertussis toxin sensitive, since control and EDG-4 transfected cellsshowed similarly low levels of IL-8 in the presence of PTX. As expected,PTX did not inhibit the IL-8 response to TPA, which is not mediated by aGPCR. Despite the presence of abundantly expressed endogenous EDG-3 RNA,COS-1 cells do not show an IL-8 response to S1P. However, heterologousexpression of rat EDG-4 reconstitutes a PTX-sensitive IL-8 response toS1P, similar to the endogenous receptor expressed in HeLa cells.Therefore, the functional assay described herein critically depends onthe expression of specific edg and/or LL receptors which are expressedendogenously in HeLa cells, and which can be heterologously expressed inthe form of EDG-4, and perhaps other related GPCRs.

EXAMPLE 10B Expression of Endogenous Edg Receptors in 293-EBNA Cells

To determine the more appropriate cells for transfection with the edgcDNA receptors, a Northern Blot experiment was conducted for HeLa, COSand 293-EBNA cells. As can be seen from FIG. 10B, the Northern blotshows that 293-EBNA cells has no visible expression of any of the edgreceptors other than possible EDG-5. In conjunction with the NorthernBlot experiment, each of these cells, HeLa, COS and 293-EBNA wereexposed to TPA, LPA and S1P and then measured for IL-8 production. The293-EBNA cells showed no IL-8 production for LPA and S1P indicating thatthere is no expression of any EDG receptor.

EXAMPLE 11 Heterologous Expression Studies Using Luciferase Assay

To improve on the 2-fold CAT reporter gene induction observed in theprevious experiment, 2 changes were made. First, the NF-κB responseelement was reconstructed in a new reporter construct (p4Luc) suitablefor stable maintenance as an episome in primate cells. Second, transienttransfection was carried out in 293-EBNA cells (Invitrogen; Cat.R620-07), an EBNA-1 expressing derivative of HEK-293. The p4-Lucreporter used the backbone of pREP4 (Invitrogen; Cat. V004-50), whichcontains the EBV origin of replication (EBV_(ori)), as well as theEBNA-1 viral antigen required to maintain EBV_(ori)-containing plasmidsas stable episomes in primate cells, and a prokaryotic selection marker.A dominant eukaryotic selection marker for zeocin resistance wassubstituted for the neo marker of pREP4, and a luciferase cassette wascloned into the multiple cloning site for expression in pREP4. Thepromoter of pREP4 was then excised and replaced with a multi-cloningsite for introduction of promoter/enhancer inserts. The NF-κB-tk insertof the previous CAT reporter was subcloned into this site and allcloning junctions were sequenced to verify the structure of the plasmid,called NF-κB-tk-p4Luc.

Assay #1

Monolayer Transient Transfection Protocol for 293-EBNA

Day 1:

1) 150 mm plates of 293-EBNA obtained from Invitrogen (Cat. R620-07)with a confluency of ˜80% were used for transfection.

2) 6.6 μg NF-κB-tk-p4Luc reporter DNA and 6.6 μg of pC3-redg4(expressing rat EDG-4), or pcDNA3 DNA was diluted in 500 μl OPTI-MEM(Gibco; Cat. 31985-062)

3) 96.8 μl Lipofectamine (Gibco; Cat. 18324-020) was diluted in 500 μlof OPTI-MEM.

4) The 2 solutions were mixed gently and the tube was incubated for 30min at room temperature.

5) The 293-EBNA plates were washed once with PBS and 13 ml OPTI-MEM wasadded to each plate.

6) 6 ml OPTI-MEM was added to each transfection tube and this was addedto a plate of 293-EBNA cells. The plates were left for 4 hrs at 37° C.in a 5% CO₂ incubator.

7) After 4 hrs, the media was removed and replaced with fresh 10% FBSmedia.

Day 2:

1) Transfected cells were washed, trypsinized with 1×trypsin,resuspended in 10 ml media and counted.

2) 0.02×10⁶ cells were plated per well of a 96-well Blackview platecoated with polyD-lysine. No cells were plated in the outside wells ofthe 96well plate. Two 96-well plates were seeded for each transfection.

Day 3:

1) Cells were washed with PBS and 140 μl serum-free media (SFM) added toeach well. Plates were incubated in 37° C. incubator for 6 hrs.

2) After 6 hrs, media was removed and cells treated with compoundsdiluted in 0.5% FBS media (140 μl added to each well).

The following treatments were used:

pcDNA3:

Untreated, LPA 10 μM, LPA 5 μM, S1P 10 μM, S1P 2 μM, SPC 3 μM, SPC 1 μM,edelfosine 1 μM, edelfosine 500 nM, LPC 1 μM, LPC 500 nM, 20% FBS(Gibco; Cat. 10437-028), TPA (50 ng/ml), TPA (25 ng/ml).

pC3-EDG-4:

Untreated, LPA 10 μM, LPA 5 μM, S1P 10 μM, S1P 5 FM, S1P 1 μM, SPC 3 μM,SPC 1 μM, edelfosine 1 μM, edelfosine 500 nM, LPC 1 μM, LPC 500 nM, 20%FBS, TPA (50 ng/ml), TPA (25 ng/ml).

3) Cells were treated for 24 hrs.

Day 4:

Luciferase Assay

1) Luclite kit (Packard; Cat. 6016911) was used for luciferase assay.All reagents were brought to room temperature before use.

2) Supernatant was transferred to a new 96-well plate and stored at −20°C. for future IL-8 measurement.

3) 50 μl 0.5M HEPES pH 7.8 buffer (1 mM MgCl₂, 1 mM CaCl₂) was added toall wells of 96-well plate. Black adhesive backing (Polyfitronics) wasaligned to the bottom of the viewplate.

4) Luclite substrate was made up by adding 10 ml substrate diluent to 1vial lyophilized substrate. Reconstituted substrate was kept under adark container. 50 μl substrate was added to each well.

5) A clear adhesive plate sealer was adjusted onto the viewplate andsealer rubbed over the plate with a Kimwipe. The plate was shaken on aplate shaker at 500 rpm for 5 seconds right side up and then upsidedown. A stop plate was placed on top of the blackview plate to keep itin the dark.

6) Plates were incubated at room temperature for 30 min.

7) After incubation, plates were counted in a 12-detector Packard TopCount on a program without dark delay.

Results: 293-EBNA cells cotransfected with pC3-redg4 and theNF-κB-tk-p4Luc reporter showed a 4-4.5-fold increase in luciferaseactivity when the cells were treated with 5 μM or 10 μM S1P (see FIG.11). EDG-4 expressing cells treated with 1 μM S1P showed a 2-foldincrease in luciferase activity. Pretreatment with PTX inhibited theresponse to S1P at all concentrations. No increase in luciferaseactivity was seen in cells cotnansfected with the empty expressionvector pcDNA3 and the luciferase reporter, and no change in luciferaseactivity was seen with PTX pretreatment in these cells. SPC also inducedthe reporter gene in EDG-4 expressing cells, but not control cells, andthis response was also PTX-sensitive. The potency of SPC was apparentlylower than that of S1P, though this was not rigorously assessed. TPAstrongly induced the NF-κB reporter, and PTX did not affect thisinduction, as expected. No induction of the reporter was seen with anyof the other ligands assayed, either in pC3-redg4 or pcDNA3-transfectedcells.

These results strongly support the assignment of EDG-4 as aPTX-sensitive S1P receptor which signals via NF-κB and inflammatory geneexpression. Furthermore, the results provide a definitive validation ofthe receptor-dependent functional assays, which comprise one aspect ofthe present invention.

The isolated receptor, which is endogenously expressed in HeLa cells,also constitutes one embodiment of the current invention. Numerousmethods well-known to those skilled in molecular biology and expressioncloning are available to isolate the edg or LL GPCR which fulfills thecriteria we have established herein. These include the screening of aHeLa cDNA library (Invitrogen; Cat. A550-26) with degenerate or specificoligonucleotides derived from EDG-4, the EDG-1/EDG-3/EDG-4 subfamily, orthe broader edg family including EDG-1 and EDG-2 paralogs, as well asscreening by hybridization with rat EDG-4 coding region DNA. Expressioncloning should also easily identify an edg/LL receptor cDNA, cloned in asuitable expression vector, which confers on 293-EBNA cells the capacityto produce IL-8 or induce a NF-κB reporter in response to S1P,SPC and/orLPA in a PTX-sensitive manner.

Assay #2

The IL-8/NF-κB response met all the criteria of a receptor-dependent,robust and reproducible functional assay of EDG/LL receptors. This assaywas applied to various cloned EDG receptors for responsiveness tonatural LL, as well as complex mixtures such as fetal bovine serum. Inthis way, agonist ligands for the orphan EDG receptors are identified,and EDG receptors which are capable of inflammatory responses areidentified.

Transient Transfection Protocol for 293-EBNA

Day 1:

The above protocol for assay 1 was followed except for the followingchanges:

1) 100 mm plates of 293-EBNA with a confluency of ˜80% were used fortransfection.

2) 3 μg NFκB-tk-p4Luc reporter DNA and 3 μg pC3-hedg1, pC3-hedg3,pC3-redg4, pC3-hedg5 or pcDNA3 DNA was diluted in 240 μl OPTI-MEM(Gibco; Cat. 31985-062)

3) 22 μl lipofectamine (Gibco; Cat. 18324-020) was diluted in 240 μlOPTI-MEM.

4) The 293-EBNA plates were washed once with PBS and 7 ml OPTI-MEM wasadded to each plate.

5) DNA/lipofectamine mixture was added to each plate of 293-EBNA cells.The plates were left for 4 hrs at 37° C. in a 5% CO₂ incubator.

Day 2:

1) 0.01×10⁶ cells were plated per well of a 96-well Blackview platecoated with polyD-lysine. No cells were plated in the outside wells ofthe 96-well plate.

Day 3:

The following treatments were used for all transfections:

Untreated, S1P 3 μM, LPA 3 μM, psychosine 3 μM (Sigma; Cat. P-9256,Stock 10 mM in methanol), SPC 3 μM, LPC 1 μM, sphingosine 3 μM, 20% FBS,TPA (20 ng/ml), edelfosine 1 μM, lysosulfatide 3 μM.

Results: 293-EBNA cells transfected with the pC3-redg4 construct showeda 3.5-fold increase in luciferase activity when the cells were treatedwith 3 μM S1P (see FIG. 12). In this experiment 3 μM SPC showed a 4-foldincrease in luciferase activity. As seen previously, PTX efficientlyinhibited the response to S1P and SPC. No response to S1P or SPC wasseen in pcDNA3-transfected 293-EBNA cells, confirming previous results.This demonstrates that the luciferase response to S1P and SPC iscritically dependent on the heterologous expression of EDG-4 in the293-EBNA cells.

Cells transfected with rat EDG-4 or human EDG-5 and treated with 20% FBSalso showed 2-fold increase in luciferase activity and PTX efficientlyinhibited this response. No such response was seen to 20% FBS inpcDNA3-transfected cells, and PTX had no effect on the luciferaseexpression of the control cells in the presence or absence of 20% FBS.S1P is present in FBS as a result of release from clotted platelets, andcan account for the increase in luciferase observed in EDG-4 expressingcells treated with 20% serum. We conclude that 20% serum contains 1 ormore agonists for EDG-5, which may consist of LPA or related LL.Moreover, EDG-5, like EDG-4, is capable of responding throughproinflammatory NF-κB signaling pathways.

These results, in addition to confirming the previous experiment,support a broad application of this robust and reproducible functionalassay in screening for agonists and antagonists of edg and LL receptors.With a positive receptor-induced readout such as IL-8 production or theNF-κB reporter gene, experiments can be carried out on transientlytransfected cells, allowing for rapid and flexible screening of a targetedg/LL receptor. This contrasts with an inhibition assay such as theG_(i)-mediated inhibition of cAMP production by forskolin. In the lattertype of assay, stable cell lines are necessary so that the decrease willnot be masked by the uninhibited response of untransfected cells.

Additionally, this approach can identify agonists for orphan edg/LLreceptors, provided the receptors respond through the inflammatorypathways described herein. Even where the natural agonist of an edgreceptor is unknown, screening for agonists is possible with theserobust and reproducible readouts. Using this approach, agonists can beidentified for heterologously (or endogenously) expressed edg/LLreceptors whether applied as chemically pure substances, ligand clips,or in biological preparations such as serum. It is a tractableproposition to purify, isolate, characterize and synthesize the activeLL from serum with this reliable bioassay in hand.

Assay #3

NE-κB activates gene expression by binding to specific DNA sequencesfound in the promoters of genes regulated by this inflammation-relatedtranscription factor. A different sequence, the serum response element(SRE) is found in the promoters of genes which are upregulated by theaddition of serum to serum-starved cells. Both LPA and S1P are found inmicromolar concentrations in serum, and have been shown to mediate asignificant part of the SRE upregulation caused by serum. Since SREactivation reflects different and distinct pathways from those leadingto NF-κB activation, EDG-4 and the closely related EDG-1 and EDG-3receptors were tested for induction of a SRE reporter gene by S1P orSPC. The SRE reporter was identical to the NF-κB reporter, except thatthe NF-κB binding sites were replaced with 2 SRE sites. The new reporterwas called 2XSREtk-p4Luc-zeo.

Transient Transfection Protocol for 293-EBNA (Assay 3):

Day 1.

The protocol described in Example 11 for Assay 1 was followed except forthe following changes:

1) 100 mm plates of 293-EBNA with a confluency of ˜80% were used fortransfection.

2) SRE Cotransfection: 0.5 μg of 2XSREtk-p4Luc-zeo reporter DNA and 3.5μg pcDNA3, EDG-1, EDG-3 (pC3-hE3HP2, different from the clone used inAssay 2 of Example 11) or the newly cloned human EDG-4 (pC3-hedg4#36);NF-κB Cotransfection: 2 μg 6XNFκBtk-p4Luc-zeo reporter DNA and 2.0 μgpcDNA3, EDG-1, EDG-3 (pC3-hE3HP2), or EDG-4 (pC3-hedg4#36). Expressionplasmid and reporter plasmid DNA samples were combined and diluted in750 μl of DMEM/F12 (serum free media) and 20 μl Plus Reagent(Lipofectamine Plus Kit, Life Technologies Cat. 10964-013), andincubated at room temperature for 15 min.

3) 30 μl Lipofectamine Reagent (Lipofectamine Plus Kit) was diluted in750 μl DMEM/F12. The diluted Lipofectamine was then combined with theDNA/Plus mixture and incubated at room temperature for 15 min.

4) The 293-EBNA plates were washed once with PBS and 5 ml DMEM/F12 wasadded to each plate.

5) DNA/Plus/Lipofectamine mixture was added to each plate of 293-EBNAcells. The plates were left for 3 hr at 37° C. in a 5% CO₂, incubator.

6) The transfection medium was replaced with serum-free DMEM/F12 forcells transfected with 2XSREtk-p4Luc-zeo reporter DNA and with DMEM/F12plus 10% FBS for cells tansfected with 6XNFκBtk-p4Luc-zeo reporter DNA.

Day 2.

2) Transfected cells were harvested by trypsinization and 50,000 cellsper well were plated in 96-well Blackview plates coated with polyD-lysine (Becton Dickinson Labware, Cat. 40640). No cells were plated inthe outside wells of the 96-well plate.

Day 3.

1) Media for cells transfected with 6XNFκBtk-p4Luc-zeo reporter DNA wasreplaced with DMEM/F12 plus 0.5%FBS.

Day 4.

1) Media was removed and cells treated with compounds diluted inDMEM/F12 media. The following treatments were used for alltransfections: Untreated: serun-free medium alone, S1P (3 μM), SPC (3μM).

2) The cells were treated for 6 hours.

3) Luciferase assay was performed.

Cotransfection of EDG-1 and 2XSREtk-p4Luc-zeo reporter resulted in a8-fold increase in luciferase activity after treatment with 3 μM S1P,and a 6-fold increase after treatment with 3 μM SPC (FIG. 13A). Incontrast, no increase in luciferase activity was seen in S1P- orSPC-treated cells cotransfected with EDG-1 and the 6XNFκBtk-p4Luc-zeoreporter (FIG. 13B). Thus, although the EDG-1 receptor is fullyfunctional, and recognizes S1P and SPC as agonists, the NF-κB reporterwas not induced. This result confirms the finding that EDG-1 is anon-inflammatory subtype of S1P/SPC receptor.

Although the original human EDG-3 clone did not produce a NF-κB responseto S1P or SPC, a different human EDG-3 clone, derived from humanpancreas (pC3-E3HP2), was cotransfected with the SRE reporter and thisclone showed a robust 12-fold SRE response to 3 μM S1P and 11-foldresponse to 3 μM SPC (FIG. 13A). A control cotransfection of the emptyexpression vector pcDNA3 with the SRE reporter showed a small butreproducible response to S1P (about 1.5-fold) but not SPC (FIG. 13A).The robust SRE response of the pancreas EDG-3 clone confirms ourhypothesis that both EDG-1 and EDG-3, in addition to the closely relatedEDG-4, function as S1P/SPC receptor subtypes. Moreover, a similarinduction of the NF-κB reporter gene (about 8-fold) was seen both inS1P- and in SPC-treated cells, compared to untreated controls, aftercotransfection with EDG-3 (FIG. 13B). No such induction was seen in thecells cotransfected with pcDNA3 and the NF-κB reporter gene (FIG. 13B),indicating that the NF-κB response to S1P and SPC in EDG-3 transfectedcells was not due to endogenous receptors. Therefore, EDG-3 (but notEDG-1) must be considered to be another edg/lysolipid receptor subtypewhich can mount an inflammatory response to S1P and otherlysosphingolipids.

Like EDG-1 and EDG-3, human EDG-4 (See Examples 12, 13 and 14 foridentification and cloning of HEDG-4) also responded through the SREreporter gene, showing a 8-fold response to S1P and a 9-fold response toSPC, relative to untreated control cells (FIG. 13A). As we hadpreviously observed with the rat EDG-4 expression construct tested inExample 11, human EDG-4 also mediated a robust NF-κB -response, showinga 4.5- and 9-fold induction of the reporter gene to S1P and SPC,respectively (FIG. 13B). Therefore, induction of inflammatory geneexpression pathways is a conserved feature of EDG-4 in humans and rats,and likely reflects a fundamental biological aspect of receptorfunction.

Together, these results suggest that the SRE response is a sharedfeature of many different edg/lysolipid receptors, and can be used toverify the response of intact, functional receptors to their cognateagonist(s). On the other hand, the NF-κB response is shared by a subsetof edg/lysolipid receptors which are specialized to mobilizeinflammatory gene expression and immune system recruitment. Since EDG-1,EDG-3, EDG-4 and EDG-7 are all S1P/SPC receptors, their varying and evenoverlapping tissue distribution and inducibility frustrate themeaningful design, screening and therapeutic testing ofanti-inflammatory S1P analogs unless the subtype specificity ofinflammatory signaling is appreciated. This complexity highlights thevalue and utility of the recombinant inflammatory lysolipid receptorsand the functional assays specified herein.

EXAMPLE 12 Identification of Human Expressed Sequence Tags (ESTs)Homologous to Rat H218 (EDG-4)

A BLAST search of the complete GenBank database was conducted with thesequence of an oligonucleotide RE4_(—)181F[5′-GAGAAGGTTCAGGAACACTACAATTACACCAA GGA-3′], based on the sequence ofrat EDG-4. The search identified a human EST (GenBank accessionAA804628), which was 88% identical to the corresponding region of ratEDG-4 cDNA (GenBank accession U10699). A subsequent TBLASTN search ofthe EST database using the predicted polypeptide product of the ratEDG-4 cDNA (according to accession number U10699) revealed 2 othermatching EST's (accession AA827835 and AA834537) in addition to theoriginal human EST. The 3 EST's encompassed the predicted translationstart site of human EDG-4 (based on similarity to rat EDG-4), overlappedeach other extensively, and together spanned some 109 codons of theN-terminal portion of the human EDG-4 polypeptide (FIG. 14). Thepredicted fragment of the human EDG-4 polypeptide showed 90.1% identityand 93.3% similarity to the equivalent fragment of rat EDG-4, suggestingthe human polypeptide is an ortholog of the rat EDG-4 gene product,rather than a closely related gene product. A BLAST search was thenconducted with the complete sequence of rat EDG-4 cDNA (accession numberU10699) against the EST database. In addition to the previouslyidentified EST's, 2 EST's apparently derived from the 3′-untranslatedregion of human EDG-4 cDNA adjacent to the poly(A) tail were found(AA767046 and N93714). Of the 5 human EST's identified in total, onlyN93714 was present in the public database before Feb. 19, 1998. This ESTwas derived from the 3′ end of a 1421 bp cDNA insert which contained nocoding region. The closest match recorded in the DBEST database entry(accession 500502) was a cGMP phosphodiesterase. The 5′ end of the clonehad been sequenced and given the GenBank accession W21101; however,similarity to other cDNAs was obscured by the presence of an Alusequence.

EXAMPLE 13 Survey of Potential cDNA Sources Using 5′ End and 3′ EndDialnostic PCR

To evaluate possible sources of human EDG-4 cDNA from HeLa cells (whichexpress the inflammatory S1P/SPC receptor) and lung (a predominant siteof EDG-4 expression in rat) for the presence of the desired cDNAfragments, diagnostic PCR primers were designed from the cluster of 5′end EST's (AA804628, AA834537 and AA827835) and 3′ end EST's (N93714 andAA767046):

5′ end primers:

HE4-DF1 [5′-ATTATACCAAGGAGACGCTGGAAAC-3′]

HE4-DR1 [5′-AGAGAGCAAGGTATTGGCTACGAAG-3′]

3′ end primers:

HE4-DF2 [5′-TCCTCTCCTCGTCACATTTCCC-3′]

HE4-DR2 [5′-GCATTCACAAGAAATTACTCTGAGGC-3′]

Template sources: 1) cDNA library from WI-38 lung fibroblasts (OrigeneTechnologies Inc., Cat. DLH-102); 2) cDNA library from human lung(Clontech, Cat. 7114-1); 3) cDNA library from HeLa cells (Invitrogen,Cat. A550-26); 4) First strand cDNA prepared in-house from HeLa celltotal RNA. Each template was amplified with each pair of primers usingthe Expand™ PCR system from Boehringer Mannheim (Cat. 1681-842).

Each reaction contained the following reagents:

2 μl 10x PCR Buffer 3 0.4 μl 25mM dNTP mix 0.6 μl Primer HE4-DF1 orHE4-DF2 (10 μM) 0.6 μl Primer HE4-DR1 or HE4-DR2(10 μM) 0.3 μl Expand ™enzyme(3 units) 15.1 μl water 1 μl cDNA template PCR conditions:Incubate: 94° C. for 2 min 30 cycles: 94° C. for 40 sec 55° C. for 1 min68° C. for 40 sec Incubate: 68° C. for 8 min Hold: 4° C.

The expected ˜200 bp 5′ PCR product was successfully amplified fromWI-38 lung cDNA (Origene), and from the first strand cDNA preparedin-house from HeLa cells. The ˜200 bp 3′ PCR product was successfullyamplified from human lung cDNA libraries (Origene and Clontech) and HeLacDNA library (Invitrogen), but not from the random hexamer-primed HeLafirst strand cDNA. Thus, the WI-38 human lung fibroblast cDNA library(Origene) appeared to be the most likely source of full length humanEDG-4 cDNA clones. More important, the successful amplification of afragment of human EDG-4 cDNA from HeLa provides a concrete demonstrationof EDG-4 expression in this S1P/SPC-responsive cell line, and directlysupports the claim of composition of matter on EDG-4 and inflammatoryS1P/SPC receptors isolated from HeLa cells. Together with fill-lengthsequence information presented below, full-length cloning and expressionof the inflammatory EDG-4 receptor from HeLa cells is reduced to asimple technical exercise for one skilled in the art.

EXAMPLE 14 Cloning of the Complete Coding Region of Human edg-4 cDNA

Two new primers were designed to amplify the complete coding region andmost of the 3′-untranslated region. The primers were based on the ESTsequences spanning the translation start site, and the EST sequencesrepresenting putative 3′-untranslated sequences of human edg-4. Providedthat these primers bind appropriately to a common template (ie. humanedg-4 cDNA), a ˜2.4 kb PCR fragment should be amplified, containing thecomplete coding region. These primers were used in a PCR reaction withthe WI-38 human lung fibroblast cDNA library (Origene) as follows:

HE4-DF3 [5′-GAGCCCCACCATGGGCAGCTTGTACT-3′]

HE4-DR2 [5′-GCATTCACAAGAAATTACTCTGAGGC-3′]

Each reaction contained the following reagents:

5 μl 10x PCR Buffer 3 l.0 μl 25 mM dNTP mix 1.5 μl Primer HE4-DF3 (10μM) 1.5 μl Primer HE4-DR2 (10 μM) 0.75 μl Expand ™ enzyme (2 units)39.25 μl water 1 μl cDNA template (250 ng or 500 ng of DNA) PCRconditions: Incubate: 94° C. for 2 min 10 cycles: 94° C. for 40 sec 60°C. for 40 sec 68° C. for 5 min 25 cycles: 94° C. for 40 sec 60° C. for40 sec 68° C. for 3 min Incubate: 68° C. for 8 min Hold: 4° C.

Amplified reactions from 250 ng (tube 227-45) and 500 ng (227-50) ofcDNA template each contained 3 PCR products 2 kb or larger. The PCRreaction and the DNA fragments from the gel were purified using QIAquickPCR purification kit (Qiagen Cat. 28106) and QIAquick gel extraction kit(Qiagen, Cat. 28704), respectively. Diagnostic PCR reactions werecarried out on each of the 3 PCR products, and all 3 yielded theexpected diagnostic PCR products using both the 5′ end and 3′ end primerpairs. Because they differed in size (˜2 kb, 2.2 and 2.4 kb) and yetamplified with primers from the translation start and the3′-untranslated region, all 3 may represent different alternativelyspliced edg-4 transcripts.

The 3 PCR products were used as templates to reamplify human edg-4 withprimers containing restriction sites suitable for cloning into anexpression vector. Two different 3′-end primers were selected withlonger (HE4-DR3) or shorter (HE4-DR4) 3′-untranslated regions. Thefollowing PCR primers and PCR conditions were used:

2 HE4-DF4 [5′-TTTAAAAAGCTTCCCACCATGGGCAGCTTGTACT-3′]

HE4-DR3 [5′-TATATATCTAGACATTCACAAGAAATTACTCTGAGGC-3′]

HE4-DR4 [5′-TATATATCTAGAGGAAATGTGACGAGGAGAGG-3′]

Each reaction contained the following reagents:

5 μl 10x PCR Buffer 3 1.0 μl 25 mM dNTP mix 1.5 μl Primer HE4-DF4 (10μM) 1.5 μl Primer HE4-DR3 or HE4-DR4 (10 μM) 0.75 μl Expand ™ enzyme (5units) 39.25 μl water 1 μl DNA PCR conditions: Incubate: 94° C. for 2min 28 cycles: 94° C. for 40 sec 60° C. for 40 sec 68° C. for 3.5 minIncubate: 68° C. for 8 min Hold: 4° C.

The amplified fragments were purified using QIAquick PCR purificationkit (Qiagen Cat. No.28106). The DNAs were restricted with HinDIII andXbaI, purified using QIAquick PCR purification kit (Qiagen Cat.No.28106) and QIAquick gel extraction kit (Qiagen, cat. no. 28704) andsubcloned into HinDIII and XbaI-restricted pcDNA3 (Invitrogen;discontinued). Sequencing was carried out using fluorescent dye-labeleddideoxy terminators and an Perkin-Elmer/ABI 377 automated sequencingapparatus, with primers designed from vector sequences flanking theedg-4 insert, or from known rat or human edgy sequence. The human edg-4sequence was compiled and assembled using the Lasergene DNAStarcomponent SeqMan. Comparisons to rat edgy were carried out with theWisconsin Group's GCG modules FRAMESEARCH, GAP, FASTA and BLAST.

A 1,170 bp span of the ˜2.4 kb human edg-4 cDNA insert was sequencedextensively. The cDNA sequence as derived from clones pC3-hedg4#5 andpC3-hedg 4#36 is presented in FIG. 15A. This region included 37 bp ofputative 5′-untranslated region, a 1059 bp open reading Same (excludingthe stop codon) corresponding to the complete human edgy coding region,and 74 bp of 3′-untranslated region adjacent to the coding region. ThiscDNA sequence showed 82.1% identity to the rat edg-4 cDNA sequence ofGenBank entry U10699 over a 1129 bp region spanning the complete openreading frames of the rat and human edg-4 polypeptides, respectively.

The predicted human edg-4 translation product (FIG. 16A) showed 90.1%identity, and 92.3% similarity to the rat EDG-4 polypeptide, consistentwith its identification as the human ortholog of rat EDG-4. An alignmentof the rat and human EDG-4 amino acid sequences is shown in FIG. 17A.The human EDG-4 polypeptide sequence has features typical of a Gprotein-coupled receptor, including 7 putative transyembrane domains,multiple potential intracellular phosphorylation sites and a singlepotential extracellular N-glycosylation site. The locations of thesefeatures are indicated in FIG. 16A.

FIGS. 15B and 16B illustrate the cDNA sequence and amino acid sequence,respectively, of the HEDG-4 receptor of clone pC3-hEdg4#36. FIG. 17Bshows the alignment of the amino acid sequences of FIGS. 16A, 16B andthe rat EDG-4.

EXAMPLE 15A S1P Activation and Functional Response of the Cloned HumanEDG-4 Receptor

To determine whether the newly identified human EDG-4 gene product, likeits rat counterpart, can respond to SPC via activation of a serumresponse element (SRE) reporter gene, the expression clone pC3-hedg4#36was transfected into 293-EBNA cells together with a luciferase reporterbearing 2 copies of a consensus binding sequence for serum responsefactor. Transfection was accomplished using the Lipofectamine Plus kit(Life Technologies, Cat. 10964-013), using the manufacturer'srecommended conditions. Optimal SRE induction was seen when cells wereseeded so as to become 100% confluent at the time of treatment, 72-96 hrafter transfection. The cells were serum-starved in medium with 0% to0.15% serum for the last 72 hr before treatment, then treated inserum-free medium for 6 hr with 3 μM SPC, or with serum-free mediumalone. Under these conditions, a control cotransfection with emptyexpression vector pcDNA3 gave about 2.5-fold induction of the SREreporter, suggesting that a low level of S1P/SPC receptor was expressedendogenously by the 293-EBNA cells. Human EDGE expression, in contrast,yielded a 26.3-fold induction of the SRE reporter gene by 3 μM SPC (FIG.18A). Similarly, rat edg4 cotransfection with the SRE reporter gave a35.6-fold induction of luciferase activity with 3 μM SPC. Thus, thehuman edg-4 cDNA encodes a functional S1P/SPC receptor, whose expressioncan be readily detected in 293-EBNA cells.

EXAMPLE 15B Determination of Relative Potency and Efficacy of HumanEDG-4 Receptor Agonists

One aspect of the present invention is a method for using recombinanthuman EDG-4 receptors in drug screening programs. Although the use ofGPCRs in high-throughput screening is well known, no such screen hasbeen reported for any edg receptor. More specifically, the novel humanEDGE receptor presented herein can be used to identify and rank therelative potency and efficacy of potential agonists. These compounds maybe useful inasmuch as they would be expected to trigger thesurvival-related signal transduction pathways associated with NF-κBinduction. Equally, once a quantitative and reliable assay isestablished, it can readily be applied to identify and rank the relativepotency and efficacy of receptor antagonists. This application, withoutlimiting other aspects, of the screening methods described herein isspecifically contemplated and incorporated within the scope of thisinvention.

Transfection of EDG-4, expression, pretreatment and treatment of293-EBNA cells expressing recombinant human EDG-4 was carried outessentially as described in “Example 11. Heterologous Expression studiesusing Luciferase Assay.” Various concentrations of S1P, SPC, psychosine,glucopsychosine or dihydrosphingosine 1-phosphate (dihydro-S1P) wereapplied in triplicate to cells in 96-well plates, and luciferase levelswere measured after 6 hr treatment. Results were tabulated in MicrosoftExcel, and analyzed with GraphPad Prism software. EC₅₀ values weredetermined using a fixed Hill-slope equation, unless variable slopesignificantly improved the fit to the data. The luciferase response wasexpressed as fold response, after subtracting any endogenous response inpcDNA3-transfected cells at a given concentration of compound. Theexperiment was repeated three times with similar results, and arepresentative experiment is shown in FIG. 18B.

Results: Table 2 summarizes the relative potency and efficacy of thecompounds tested. Compound EC₅₀ (μM) Rank Max. Fold E_(Max) (Percent)Rank S1P 0.32 1 5.60 86.7 2 SPC 0.88 3 5.77 100 1 Psychosine^(a) >10 41.78 30.9 5 Glucospychosine^(a) >10 4 1.81 31.4 4 Dihydro-S1P 0.53 22.84 49.2 3 ^(a)Cytotoxicity was seen at 10 μM or higher concentrations,preventing quantitative determination of EC₅₀ or E_(Max)

Results: From the results obtained here, it can be concluded that EDG-4responds to both S1P and SPC as full agonists with similar potency andefficacy. In contrast, dihydro-S1P was a partial agonist under theseassay conditions, despite an apparent potency similar to S1P and SPC.Thus, while the addition of a choline substituent to the phosphateheadgroup did not greatly affect activity, the unsaturated carbon-carbonbond appears to play a role for full agonist activity. Psychosine andglucopsychosine both showed poor potency and efficacy, as well ascytotoxicity at higher concentrations. Nonetheless, these compounds didactivate the receptor (since pcDNA3 activity was set to 1.0 at eachconcentration). Published literature supports the existence of multiplereceptors for S1P, and the identity of at least some of these with SPCreceptor subtypes.

EXAMPLE 16 Role of Inflammatory Iysolipid Receptors in Nerve GrowthFactor-mediated Inflammation and Neurotronhic Signal Transduction

The use of sphingosine 1-phosphate (S1P) in suppressing programmed celldeath is known (Cuvillier et al., 1996; Spiegel, 1998). However, sinceS1P was presumed to act as an intracellular second messenger, noreceptor-based data were presented. Our own work shows that the Gprotein-coupled receptors (GPCRs) EDG-1 (Hla & Maciag, 1990), EDG-3(Yamaguchi et al., 1996), EDGA-4 (referred to in published literature asAGR16 [Okazaki et al., 1993] or H218 [MacLennan et al., 1994]) andHEDG-4 as cloned herein, and EDG-7 (Munroe et al., unpublished;corresponding U.S. Ser. No. 60/070,185, incorporated herein byreference) respond to S1P and sphingosylphosphorylcholine(SPC) as anagonist. However, as shown in the previous examples and in Example 18below, only two of the four S1P/SPC receptors signal through activationof NF-κB: EDG-3 and EDG-4. S1P has multiple biological activitiesincluding mitogenesis, neurite retraction, inhibition of cell motility,suppression of apoptosis and as we have found, inflammatory geneexpression. Therefore, successful therapeutic use of S1P or its analogshinges on recognizing which receptors are expressed, and what theirfunction(s) are in tissues exposed to the agent.

Direct modulation of NF-κB activation cascades has been proposed as atherapeutic mechanism for inflammation or apoptosis. However, NF-κBplays a vital role in innate immunity against ubiquitous microbialpathogens and in mobilizing the antigen-specific immune system.Therefore, rather than targeting this irreplaceable defense system, itwould be preferred to instead block inappropriate activation of NF-κBthrough inflammatory S1P/SPC receptors, in situations where theiragonists and/or receptor signaling are excessive or inappropriate.Alternatively, where NF-κB could prevent unwanted apoptosis or couldenhance immune function in immunocompromised hosts, agonists of thesereceptors would be desirable, especially with favorable medicinalchemistry properties and selective pharmacology.

Because the sphingosine-phosphorylating enzyme sphingosine kinase(Edsall et al., 1997) and NF-κB (Rius et al., 1997) have both been shownto play critical roles in the neurotrophic action of NGF in thewell-defined PC12 neuroblastoma model, we can surmise that theanti-apoptotic signaling pathway of NGF depends on both S1P and NF-κB.EDG-4 has been shown to be expressed in PC12 cells before, during andafter NGF treatment (MacLennan et al., 1994). In CNS, the highest levelsof edg-4 RNA are detected during embryogenesis. Immunohistochemicallocalization of CNS EDG-4 protein labels cell bodies and axons to young,differentiating neurons, consistent with the proposed role inneurotrophic function (MacLennan et al., 1997).

Since EDG-4 responds to S1P/SPC by activating NF-κB, it can be predictedthat a causal link between S1P production (Edsall et al., 1997) andNF-κB activity (Rius et al., 1997) exists in PC12 cells. EDG-3, ifexpressed, could play a similar role. Although many steps in NGFsignaling have been described, no report exists which links S1P to NF-κBin this system. In U937 cells, a single report does show that S1Ptreatment resulted in NF-κB activation (Shatrov et al., 1997). However,the authors did not show whether inflammatory gene expression such asIL-8 or IL-6 resulted, nor did they realize that a cell-surface receptorcould be involved. Instead they assumed that S1P is an intracellularsecond messenger, as indeed did U.S. Pat. No. 5,712,262 (Cuvillier etal., 1996; Spiegel, 1998). We have now provided a molecular explanationof the link between these signaling steps. S1P acts on an inflammatoryreceptor subtype such as EDG-4 or EDG-3. This in turn leads to theactivation of the G_(i/o) heterotrmeric protein complex, triggeringdownstream events that depend on tyrosine kinase(s) and reactive oxygenspecies. Finally, NF-κB is activated, resulting in anti-apoptotic geneexpression.

Two receptors exist for NGF on PC12 cells and many other neuronal andnon-neuronal cell types. One of these, TrkA, is a high-affmity NGFreceptor which signals through a classical dimeric transmembranetyrosine kinase receptor mechanism. The other, p75^(NGFR), is a lowaffinity receptor for NGF and several other neurotrophins, belongs tothe “death receptor” gene family including TNFR, Fas/CD95 and CD28, andsignals through a sphingomyelinase pathway using ceramide and/orsphingosine as key pro-apoptotic intermediates. In fact, p75^(NGFR)expression in the absence of TrkA causes NGF to induce apoptosis, ratherthan survival of PC12 cells. TrkA co-expression with p75^(NGFR) isrequired for NGF to display neurotrophic activity in PC12 cells;expression of TrkA alone is without effect on apoptosis.

Without wishing to be bound by theory, it appears that TrkA confersneurotrophic activity on NGF as follows. Sphingosine kinase (SK) is anenzyme that converts the proapoptotic sphingosine into S1P. S1P has beenshown to actively suppress programmed cell death induced by deathreceptor ligands or ceramide (Cuvillier et al., 1996; Spiegel, 1998). SKis induced by NGF in PC12 cells that co-express TrkA and p75NGFR, butnot when the tyrosine kinase activity of TrkA is inhibited with K252a(Edsall et al., 1997). Therefore, it appears that the induction ofsphingosine kinase converts a p75^(NGFR) death signal(ceramnide/sphingosine) into a survival signal (S1P). Given the presenceof EDG-4 (and perhaps EDG-3) in PC12 cells, the production of S1P viasphingosine kinase would be expected to lead to activation of the GPCR,thereby activating NF-κB. NF-κB, in turn, is already known to beessential for neurotrophic responses to NGF (Rius et al., 1997). Thus,inflammatory S1P receptors play a pivotal role in directly linking thesetwo essential steps in NGF neurotrophic signaling.

Like p75^(NGFR), several other death receptors have been shown to induceapoptosis and/or NF-κB activation, depending on the cell type andcostimulus applied. The involvement of sphingomyelinase,ceramide/sphingosine and sphingosine kinase in the signaling cascade hasalso been shown repeatedly with TNFR, Fas/CD95 and other family members.Another parallel with the NGF system is the observation that some celltypes that express a given death receptor survive their ligands whileother do not. Again, protein kinase C is implicated in survivalpathways. There is even direct evidence that S1P plays a similar role insurvival for Fas/CD95 and in inflammatory gene expression for TNFR.Therefore, one can predict a widespread role for inflammatorylysosphingolipid/edg receptors in modulating the apoptotic/inflammatorypotential of death receptor ligands. If true, these GPCRs may play afundamental role in cell survival, differentiation, and inflammation.Therefore, methods for isolating such receptors, and for identifyingligands that modulate these activities constitute aspects of theinvention described herein.

The ligands for other GPCRs known to activate NF-κB are generallypeptides or small molecules produced in a very limited range of celltypes. However, the sphingolipids and sphingomyelinase which areubiquitously distributed can be used to generate ligands for the edgreceptors. Therefore, potentially every cell type can make ligands forthese receptors. Moreover, ceramide and/or sphingosine are synthesizedas an integral part of the death receptor signaling pathways, so thatsurvival may require as little as a single additional metabolicconversion to S1P, provided the appropriate S1P receptors are present.While TrkA provides the signal to induce SK in PC12 cells, otherinducers of protein kinase C have also been shown to induce SKexpression. One of these is the potent tumor promoter phorbol ester.Thus, other costimulators may dramatically change or even reverse theoutcome of death receptor signaling through the inflammatory S1P/SPCreceptors.

Screening of individual S1P/SPC receptors will permit the identificationand optimization of selective ligands for use in modulating apoptosisand inflammation. For example, SPC shows greater activity than S1Pacting on EDG-4, whereas the 2 compounds have similar activity on theEDG-3 receptor. While anti-apoptotic compounds directed at these targetsare difficult to identify without the receptor assays, selectivepro-apoptotic compounds are even harder to target, since many enzymeinhibitors can trigger apoptotic pathways. Furthermore, since it nowappears that edg receptor-induced NF-κB is one mechanism by which S1Psuppresses apoptosis, inflammatory gene expression is also expected tooccur. A further implication is the potential for immune stimulationwith EDG-3 or EDGY agonists, including S1P and SPC. Antagonists, on theother hand, could be used to treat transplant rejection or autoimmunediseases, in which both inflammatory responses and insufficientapoptosis of auto/alloreactive T cells play a role.

EXAMPLE 17 Three Inflammatory Subtypes of Lysophosphatidic Acid (LPA)Receptor

LPA, like S1P, is abundant in serun, but not plasma. Moreover, LPA isproduced as a consequence of phospholipase A₂ with or without thecontribution of phospholipase D (depending on the phospholipidsubstrate). Our results showing IL-8 production in HUVEC exposed to 5 μMLPA further suggest that inflammatory responses could be mediated by.some, or all, LPA receptors. To date we have identified three subtypesof edg receptors that respond to LPA as an agonist. These are EDG-2,EDG-6 and EDG-5 (referred to also as LP_(A1), LP_(A2) and LP_(A3),respectively (Chun, J, Contos, J J A and Munroe, D G. 1998. A growingfamily of receptor genes for lysophosphatidic acid (LPA) and otherlyso-phospholipids. Cell Biochem Biophys (in press)). The EDG-5 receptoris set out in co-pending U.S. application Ser. No. 08/997,803 to MLUNROEet al., incorporated herein by reference and the amino acid sequence andcDNA sequence for the EDG-6 receptor is set out in FIGS. 21 and 22,respectively. To determine whether these receptors might mediateinflammatory responses, each was cotransfected separately with SRE,NF-κB or AP-1 reporter genes. The AP-1 reporter contained approximately1 kb of the human collagenase II promoter, and the first 50 bp of the5′-untranslated region of the collagenase II transcription unit(Angel P,et al. 1987. Phorbol ester-inducible genes contain a common cis elementrecognized by a TPA-modulated trans-acting factor. Cell 49:729-739), aregion whose inducible expression has been shown to be controlled byAP-1. This transcription factor, like NF-κB has been implicated ininflammatory and neoplastic signal transduction., though the genetargets of its action are largely distinct from those of NF-κB (AdcockIM. 1997. Transcription factors as activators of gene transcription:AP-1 and NF-κB. Monaldi Arch Chest Dis 52:178-186. Review).

293-EBNA cells were grown, lipofected in monolayer cultures, andpretreated as described above for Example 11, assay #1, except thatNF-κB and AP-1 reporter-transfected cells were pretreated for 6 hr inmedium containing 0.5% FBS, then treated overnight in the same mediumwith or without 10 μM LPA.

Results: As shown in FIG. 23, all three receptors robustly activated theNF-κB reporter (about 3-4-fold) in the presence of 10 μM LPA, while noresponse to LPA was seen when the NF-κB reporter was cotransfected withthe empty expression vector pcDNA3. With the SRE and AP-1 reportergenes, some endogenous response to LPA was seen (about 1.5-fold vsuntreated control cells). However, EDG-6 strongly induced bothreporters, while EDG-2 and EDG-5 caused greater than 2-fold induction ofthe SRE and AP-1 reporters with LPA. Therefore, all three LPA receptorstested here are capable of inducing inflammatory gene transcriptionthrough NF-κB , and perhaps, AP-1 as well. As mentioned, these twoinflammatory transcription factors respond to different signalingpathways by inducing distinct gene sets. However, some genes arepowerfully and synergistically activated by both factors acting inconcert (Stein B, et al. 1993. Cross-coupling of the NF-κB p65 andFos/Jun transcription factors produces potentiated biological function.EMBO J 12:3879-3891). Thus, the LPA receptors EDG-2, EDG-5 and EDG-6 arelikely to respond to LPA or other lysolipid agonists by activating oneor both sets of gene targets controlled by NF-κB and AP-1. Sincephospholipase action and NF-κB/AP-1 activation are common features ofmany diseases with an inflammatory or immune component, it is alsopossible that edg/LPA receptors exacerbate a pre-existing disease orinjury through their inflammatory responses to lysolipids. Therefore,antagonists of one or more of these inflanmnatory receptors could beuseful in treating such diseases. Without limiting the intended scope ofthe inventions disclosed, examples include rheumatoid arthritis, stroke,neurotrauma, Alzheimer's disease, ALS, asthma, endotoxic shock,atherosclerosis and many other diseases. Besides inflammation,activation of NF-κB is likely to promote survival in the face ofpro-apoptotic signals, for example, those initiated by the TNF receptorsor other “death receptors”. (Van Antwerp D J, et al. 1998. Inhibition ofTNF-induced apoptosis by NF-κB. Review. Trends Cell Biol 8:107-111) Thismay explain the observed reduction in efficacy of chemotherapy-inducedapoptosis in LPA-treated ovarian cancer cells.(Frankel A, et al. 1996.Peptide and lipid growth factors decreasecis-diamminedichloroplatinum-induced cell death in human ovarian cancercells. Clin Cancer Res 2:1307-1313) With the present disclosure,antagonists of inflammatory LPA receptors may be discovered andoptimized to reduce or delay the emergence of cancer cell populationsimmune to the apoptosis-inducing effects of chemotherapeutics. Suchtherapies may also be used to treat autoimmunity or other diseases whereexcessive or inappropriate cell survival occurs. Alternatively, agonistsof inflammatory LPA receptors may be neuroprotective, or promotesurvival of other cell types in diseases where inappropriate orexcessive cell death occurs. Examples include HIV/AIDS, myelodysplasia,endotoxic shock, cirrhosis of the liver, to name a few.

EXAMPLE 18 Calcium Microfluorimetry as a Real-time Readout of EDGReceptor Functional Responses

Reporter gene assays, while very useful, produce an endpoint assayresult, and therefore cannot give information about transient,reversible or desensitizing responses initiated by EDG receptors.Calcium microfluorimetry is one example of an alternative approach thatdoes allow such information to be gathered. Since Ca²⁺ responses to S1Por LPA have been observed in cells that endogenously express theirreceptors (Tomquist K, et al. 1997. Sphingosine 1-phosphate mobilizessequestered calcium, activates calcium entry, and stimulatesdeoxyribonucleic acid synthesis in thyroid FRTL-5 cells. Endocrinology138:4049-4057; Holtsberg F W, et al. 1997. Lysophosphatidic acid inducesa sustained elevation of neuronal intracellular calcium. J Neurochem.69:68-75) we tested 293-EBNA cells transiently transfected withdifferent EDG receptors for functional responses via calciummicrofluorimetry.

Transfections were carried out with EDG receptors in 293-EBNA cells asdescribed above, except that no reporter gene vector was included in theDNA mix. Two days after transfection, cells were harvested bytrypsinization and plated at a density of 200,000 cells ontopoly-D-lysine-coated coverslips in 100 μl of medium containing 0.5% FBS.After briefly allowing cell attachment to take place, 2 ml of mediumwithout FBS was added and the cells were incubated overnight. The nextday, cells were loaded with 5 μM fura-2 AM ester (Molecular Probes) for60 min at RT, then washed and used for calcium microfluorimetry. S1P wasprepared as a 10 mM stock in 100% ethanol and diluted to a finalconcentration of 2 μM in ACSF; PMA was used at a final concentration of25 ng/ml. Treatments were applied using a gravity-fed perfusionapparatus. Fluorescence emission was continuously monitored and recordedwith PTI 2.060a software and analyzed with Sigma Plot software.Intracellular calcium concentrations were calculated by interpolation ona ratiometric fluorescence curve generated from fura-2 fluorescence in acalcium dilution series.

Results: FIG. 19 shows the response of control cells transfected withpcDNA3 and treated with 2 μM S1P. A small increase in intracellularcalcium concentration was observed with 2 μM S1P, and this responsecompletely desensitized the response to a second application of S1P.FIG. 20 shows the calcium response to S1P in EDG-3 transfected cells. Incontrast to the approximantely 60 mM change in intracellular calcium inpcDNA3-transfected cells, a 300 nM increase was observed in EDG-3transfected cells treated with 2 μM S1P. A second application of S1Pelicited a small response, though desensitization clearly occurred. TheTable below shows a qualitative analysis of preliminary data we haveobtained from cells expressing each EDG receptor, after addition of theappropriate agonist at a 2 or 10 μM concentration.

TABLE 3 Qualititative calcium response of EDG-transfected cells toreceptor agonists. Receptor Agonist Concentration Response EDG-1 S1P 2and 10 μM None within 20 min EDG-2 LPA 10 μM ++ EDG-3 S1P 2 and 10 μM+++ EDG-4 S1P 2 and 10 μM +++ EDG-5 LPA 2 and 10 μM +++ EDG-6 LPA 2 and10 μM +++ EDG-7 S1P 2 and 10 μM None within 20 min

While further experiments are required to quantitatively assess thecapacity of these receptor subtypes to elevate intracellular calcium,initial results strongly suggest a correlation of calcium signaling withinduction of inflammatory response pathways. Supporting this conclusion,EDG-1 and EDG-7 both respond through the SRE reporter to S1P, yet failto signal through NF-κB reporters or increases in intracellular calcium.The fact that only two of the four identified S1P receptors signalthrough NF-κB indicates that effective anti-inflammatory orsurvival-modulating therapeutics can best be developed using theinventions disclosed herein, which specifically measure the relevantreceptor subtypes and pathways as indicators of therapeutic efficacy.Therefore, NF-κB reporter genes, other endpoint assays that measureinflammatory signal transduction or gene expression, and real-timefunctional assays that monitor inflammatory signaling by edg/LLreceptors are specifically encompassed within the scope of the presentinvention.

EXAMPLE 19 Construction and Functional Testing of a Human EDG-4 FusionProtein with Jellyfish Green Fluorescent Protein (GFP)

Chimeric proteins may be used to study the structure, function,mechanism of activation or biological role of a protein. In the case ofedg receptors, little is known of their intracellular trafficking,post-translational processing, or physical interaction with otherproteins. The green fluorescent protein (GFP) from Aequorea victoria hasbeen used as a tool for the direct visualization of various fusionproteins in living cells, since no fixation or substrate addition isrequired to obtain fluorescence. Numerous examples exist of differentproteins that retain function after fusion to GFP, including at leastsome GPCRs. (Kallal L, et al. 1998. Visualization of agonist-inducedsequestration and down-regulation of a green fluorescent protein-taggedbeta2-adrenergic receptor. J Biol Chem 273:322-328). To address ofquestions of EDG-4 trafficking and protein-protein interactions, weconstructed a GFP fusion with human EDG-4 cDNA and tested for afunctional response to S1P using the SRE reporter gene as a readout.

A pair of primers was designed from two ends of reading frame of humanedg-4 cDNA sequence to engineer the edg-4 open reading frame into avector designed for GFP fusion protein expression, with the GFP tagcarboxy-terminal to the full-length EDG-4 polypeptide:

5′-End Primer: Contains Site for Kpn I enzyme, and optimized (Kozak)translation initiation sequence:

HE4-ATG KpnF: [5′-TTTAAAGGTACCGCCACCATGGGCAGCTTGTAC-3′]

3′-End Primer: Contains site for XbaI enzyme, and lacksnaturally-occurring edg-4 stop codon:

HE4-xba/1096R: [5′-TATATATCTAGAGACCACCGTGTTGCCCTCCAG-3′]

pc3-hedg4#36 plasmid DNA was amplified with the above pair of primersunder the following conditions of PCR amplification, using the Expand™PCR system from Boehringer Mannheim (Cat. 1681-842).

The reaction contained the following reagents:

5 μl of 10× PCR Buffer 3

1.0 μl of 25 mM dNTP mix

1.5 μl of Primer HE4-ATG KpnF (10 μM)

1.5 μl of Primer HE4-xba/1096R (10 μM)

0.75 μl of Enzyme (2 units)

39.25 μl water

1μl DNA

PCR conditions: Incubate: 94° C. for 2 min 10 cycles: 94° C. for 1 min50° C. for 1 min 68° C. for 2 min 20 cycles: 94° C. for 1 min 68° C. for3 min Incubate: 68° C. for 8 min Hold: 4° C.

The amplified reaction (designated as sample 80727-3) was purified usingQIAquick PCR purification kit (Qiagen Cat.28106). The DNA was restrictedwith KpnI and Xba I enzymes, and subcloned into Kpn I and XbaIrestricted pcDNA3.1/CT-GFP (Invitrogen, Cat K4820-01). Three positiveclones i.e. E4-GFP#8-3, E4-GFP#15-3, E4-GFP#17-3 were identified,sequenced to confirm the expected insert and cloning junction, andtested by lipofection into 293-EBNA cells as described above for humanedg-4 cDNA.

Results: Cells were observed under fluorescence microscopy using afluorescein filter set. Cells expressing the EDG-4/GFP fusion proteinwere easily identified due to their bright green fluorescence. Inuntreated, serum-starved cells most of the fluorescence was peripherallylocated, apparently at the plasma membrane. However, 72 hr aftertransfection, high levels of the GFP fusion protein accumulated indiscrete clusters which might be “capped” on the cell surface or,alternatively, internalized in vesicles. A control transfection with anonfusion GFP construct revealed only a diffuse cytoplasmic localizationof GFP fluorescence. Importantly, the EDG-4/GFP receptors could bedirectly visualized in living cells without special fixing ordevelopment. Thus, trafficking and interaction of EDG-4/GFP with variousorganelles may be followed in living cells before, during and afteraddition of agonists and/or pharmacological treatments. Suchlocalization would only be meaningful, of course, if the receptors bindligands and activate signal transduction pathways normally. Results ofSRE reporter gene cotransfection and response to 1 or 5 μM S1P are shownin FIG. 24. All 3 clones of EDG-4/GFP did not differ significantly fromthe EDG-4 parent is expression vector in SRE response to S1P. Thus,despite the fairly large fusion domain presented by GFP, apparentlynormal ligand-responsiveness and intracellular signaling was retained.Visualization and quantitation of fusion receptor internalization offersan alternative means of assessing functional activation of the EDG-4receptor, for example, in pharmacological evaluation of partial agonistsof EDG-4.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments.

References

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Carter, B D, Kaltschmidt, C, Kaltschmidt, B, Offenhauser, N,Bohm-Matthaei, R, Baeuerle, P and Barde, Y-A. 1996. Selective activationof NF-κB by nerve growth factor through the neurotrophin receptor p75.Science 272: 542-545.

Cuvillier, O, Pirianov, G, Kleuser, B, Vanek, P G, Coso, O A, Gutkind, JS and Spiegel, S. 1996. Suppression of programmed cell death bysphingosine-1-phosphate. Nature 381: 800-803.

Cuvillier, O, Rosenthal, D S, Smulson, M E and Spiegel, S. 1998.Sphingosine 1-phosphate inhibits activation of caspases that cleavepoly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediatedapoptosis in Jurkat T lymphocytes. J Biol Chem 273: 2910-2916.

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MacLennan, A J, Browe, C S, Gaskin, A A, Lado, D C and Shaw, G. 1994.Cloning and characterization of a putative G-protein coupled receptorpotentially involved in development. Mol Cell Neurosci 5: 201-209.

MacLennan, A J, Marks, L, Gaskin, A A and Lee, N. 1997. Embryonicexpression pattern of H218, a G-protein coupled receptor homolog,suggests roles in early mammalian nervous system development.Neuroscience 79: 217-224.

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14 1 1170 DNA Homo sapiens CDS (38)..(1099) 1 aaagccccat ggccccagcaggcctctgag ccccacc atg ggc agc ttg tac tcg 55 Met Gly Ser Leu Tyr Ser 15 gag tac ctg aac ccc aac aag gtc cag gaa cac tat aat tat acc aag 103Glu Tyr Leu Asn Pro Asn Lys Val Gln Glu His Tyr Asn Tyr Thr Lys 10 15 20gag acg ctg gaa acg cag gag acg acc tcc cgc cag gtg gcc tcg gcc 151 GluThr Leu Glu Thr Gln Glu Thr Thr Ser Arg Gln Val Ala Ser Ala 25 30 35 ttcatc gtc atc ctc tgt tgc gcc att gtg gtg gaa aac ctt ctg gtg 199 Phe IleVal Ile Leu Cys Cys Ala Ile Val Val Glu Asn Leu Leu Val 40 45 50 ctc attgcg gtg gcc cga aac agc aag ttc cac tcg gca atg tac ctg 247 Leu Ile AlaVal Ala Arg Asn Ser Lys Phe His Ser Ala Met Tyr Leu 55 60 65 70 ttt ctgggc aac ctg gcc gcc tcc gat cta ctg gca ggc gtg gcc ttc 295 Phe Leu GlyAsn Leu Ala Ala Ser Asp Leu Leu Ala Gly Val Ala Phe 75 80 85 gta gcc aatacc ttg ctc tct ggc tct gtc acg ctg agg ctg acg cct 343 Val Ala Asn ThrLeu Leu Ser Gly Ser Val Thr Leu Arg Leu Thr Pro 90 95 100 gtg cag tggttt gcc cgg gag ggc tct gcc ttc atc acg ctc tcg gcc 391 Val Gln Trp PheAla Arg Glu Gly Ser Ala Phe Ile Thr Leu Ser Ala 105 110 115 tct gtc ttcagc ctc ctg gcc atc gcc att gag cgc cac gtg gcc att 439 Ser Val Phe SerLeu Leu Ala Ile Ala Ile Glu Arg His Val Ala Ile 120 125 130 gcc aag gtcaag ctg tat ggc agc gac aag agc tgc cgc atg ctt ctg 487 Ala Lys Val LysLeu Tyr Gly Ser Asp Lys Ser Cys Arg Met Leu Leu 135 140 145 150 ctc atcggg gcc tcg tgg ctc atc tcg ctg gtc ctc ggt ggc ctg ccc 535 Leu Ile GlyAla Ser Trp Leu Ile Ser Leu Val Leu Gly Gly Leu Pro 155 160 165 atc cttggc tgg aac tgc ctg ggc cac ctc gag gcc tgc tcc act gtc 583 Ile Leu GlyTrp Asn Cys Leu Gly His Leu Glu Ala Cys Ser Thr Val 170 175 180 ctg cctctc tac gcc aag cat tat gtg ctg tgc gtg gtg acc atc ttc 631 Leu Pro LeuTyr Ala Lys His Tyr Val Leu Cys Val Val Thr Ile Phe 185 190 195 tcc atcatc ctg ttg gcc atc gtg gcc ctg tac gtg cgc atc tac tgc 679 Ser Ile IleLeu Leu Ala Ile Val Ala Leu Tyr Val Arg Ile Tyr Cys 200 205 210 gtg gtccgc tca agc cac gct gac atg gcc gcc ccg cag acg cta gcc 727 Val Val ArgSer Ser His Ala Asp Met Ala Ala Pro Gln Thr Leu Ala 215 220 225 230 ctgctc aag acg gtc acc atc gtg cta ggc gtc ttt atc gtc tgc tgg 775 Leu LeuLys Thr Val Thr Ile Val Leu Gly Val Phe Ile Val Cys Trp 235 240 245 ctgccc gcc ttc agc atc ctc ctt ctg gac tat gcc tgt ccc gtc cac 823 Leu ProAla Phe Ser Ile Leu Leu Leu Asp Tyr Ala Cys Pro Val His 250 255 260 tcctgc ccg atc ctc tac aaa gcc cac tac ytt ttc gcc gtc tcc acc 871 Ser CysPro Ile Leu Tyr Lys Ala His Tyr Xaa Phe Ala Val Ser Thr 265 270 275 ctgaat tcc ctg ctc aac ccc gtc atc tac acg tgg cgc agc cgg gac 919 Leu AsnSer Leu Leu Asn Pro Val Ile Tyr Thr Trp Arg Ser Arg Asp 280 285 290 ctgcgg cgg gag gtg ctt cgg ccg ctg cag tgc tgg cgg ccg ggg gtg 967 Leu ArgArg Glu Val Leu Arg Pro Leu Gln Cys Trp Arg Pro Gly Val 295 300 305 310ggg gtg caa gga cgg agg cgg ggc ggg acc ccg ggc cac cac ctc ctg 1015 GlyVal Gln Gly Arg Arg Arg Gly Gly Thr Pro Gly His His Leu Leu 315 320 325cca ctc cgc agc tcc agc tcc ctg gag agg ggc atg cac atg ccc acg 1063 ProLeu Arg Ser Ser Ser Ser Leu Glu Arg Gly Met His Met Pro Thr 330 335 340tca ccc acg ttt ctg gag ggc aac acg gtg gtc tga gggtgggggt 1109 Ser ProThr Phe Leu Glu Gly Asn Thr Val Val 345 350 ggaccaacaa ccaggccagggcataggggt tcatggaaag gccactgggt gaccccaaat 1169 a 1170 2 353 PRT Homosapiens misc_feature (273)..(273) Unknown Xaa refers to any amino acid 2Met Gly Ser Leu Tyr Ser Glu Tyr Leu Asn Pro Asn Lys Val Gln Glu 1 5 1015 His Tyr Asn Tyr Thr Lys Glu Thr Leu Glu Thr Gln Glu Thr Thr Ser 20 2530 Arg Gln Val Ala Ser Ala Phe Ile Val Ile Leu Cys Cys Ala Ile Val 35 4045 Val Glu Asn Leu Leu Val Leu Ile Ala Val Ala Arg Asn Ser Lys Phe 50 5560 His Ser Ala Met Tyr Leu Phe Leu Gly Asn Leu Ala Ala Ser Asp Leu 65 7075 80 Leu Ala Gly Val Ala Phe Val Ala Asn Thr Leu Leu Ser Gly Ser Val 8590 95 Thr Leu Arg Leu Thr Pro Val Gln Trp Phe Ala Arg Glu Gly Ser Ala100 105 110 Phe Ile Thr Leu Ser Ala Ser Val Phe Ser Leu Leu Ala Ile AlaIle 115 120 125 Glu Arg His Val Ala Ile Ala Lys Val Lys Leu Tyr Gly SerAsp Lys 130 135 140 Ser Cys Arg Met Leu Leu Leu Ile Gly Ala Ser Trp LeuIle Ser Leu 145 150 155 160 Val Leu Gly Gly Leu Pro Ile Leu Gly Trp AsnCys Leu Gly His Leu 165 170 175 Glu Ala Cys Ser Thr Val Leu Pro Leu TyrAla Lys His Tyr Val Leu 180 185 190 Cys Val Val Thr Ile Phe Ser Ile IleLeu Leu Ala Ile Val Ala Leu 195 200 205 Tyr Val Arg Ile Tyr Cys Val ValArg Ser Ser His Ala Asp Met Ala 210 215 220 Ala Pro Gln Thr Leu Ala LeuLeu Lys Thr Val Thr Ile Val Leu Gly 225 230 235 240 Val Phe Ile Val CysTrp Leu Pro Ala Phe Ser Ile Leu Leu Leu Asp 245 250 255 Tyr Ala Cys ProVal His Ser Cys Pro Ile Leu Tyr Lys Ala His Tyr 260 265 270 Xaa Phe AlaVal Ser Thr Leu Asn Ser Leu Leu Asn Pro Val Ile Tyr 275 280 285 Thr TrpArg Ser Arg Asp Leu Arg Arg Glu Val Leu Arg Pro Leu Gln 290 295 300 CysTrp Arg Pro Gly Val Gly Val Gln Gly Arg Arg Arg Gly Gly Thr 305 310 315320 Pro Gly His His Leu Leu Pro Leu Arg Ser Ser Ser Ser Leu Glu Arg 325330 335 Gly Met His Met Pro Thr Ser Pro Thr Phe Leu Glu Gly Asn Thr Val340 345 350 Val 3 1062 DNA Homo sapiens CDS (1)..(1062) 3 atg ggc agcttg tac tcg gag tac ctg aac ccc aac aag gtc cag gaa 48 Met Gly Ser LeuTyr Ser Glu Tyr Leu Asn Pro Asn Lys Val Gln Glu 1 5 10 15 cac tat aattat acc aag gag acg ctg gaa acg cag gag acg acc tcc 96 His Tyr Asn TyrThr Lys Glu Thr Leu Glu Thr Gln Glu Thr Thr Ser 20 25 30 cgc cag gtg gcctcg gcc ttc atc gtc atc ctc tgt tgc gcc att gtg 144 Arg Gln Val Ala SerAla Phe Ile Val Ile Leu Cys Cys Ala Ile Val 35 40 45 gtg gaa aac ctt ctggtg ctc att gcg gtg gcc cga aac agc aag ttc 192 Val Glu Asn Leu Leu ValLeu Ile Ala Val Ala Arg Asn Ser Lys Phe 50 55 60 cac tcg gca atg tac ctgttt ctg ggc aac ctg gcc gcc tcc gat cta 240 His Ser Ala Met Tyr Leu PheLeu Gly Asn Leu Ala Ala Ser Asp Leu 65 70 75 80 ctg gca ggc gtg gcc ttcgta gcc aat acc ttg ctc tct ggc tct gtc 288 Leu Ala Gly Val Ala Phe ValAla Asn Thr Leu Leu Ser Gly Ser Val 85 90 95 acg ctg agg ctg acg cct gtgcag tgg ttt gcc cgg gag ggc tct gcc 336 Thr Leu Arg Leu Thr Pro Val GlnTrp Phe Ala Arg Glu Gly Ser Ala 100 105 110 ttc atc acg ctc tcg gcc tctgtc ttc agc ctc ctg gcc atc gcc att 384 Phe Ile Thr Leu Ser Ala Ser ValPhe Ser Leu Leu Ala Ile Ala Ile 115 120 125 gag cgc cac gtg gcc att gccaag gtc aag ctg tat ggc agc gac aag 432 Glu Arg His Val Ala Ile Ala LysVal Lys Leu Tyr Gly Ser Asp Lys 130 135 140 agc tgc cgc atg ctt ctg ctcatc ggg gcc tcg tgg ctc atc tcg ctg 480 Ser Cys Arg Met Leu Leu Leu IleGly Ala Ser Trp Leu Ile Ser Leu 145 150 155 160 gtc ctc ggt ggc ctg cccatc ctt ggc tgg aac tgc ctg ggc cac ctc 528 Val Leu Gly Gly Leu Pro IleLeu Gly Trp Asn Cys Leu Gly His Leu 165 170 175 gag gcc tgc tcc act gtcctg cct ctc tac gcc aag cat tat gtg ctg 576 Glu Ala Cys Ser Thr Val LeuPro Leu Tyr Ala Lys His Tyr Val Leu 180 185 190 tgc gtg gtg acc atc ttctcc atc atc ctg ttg gcc gtc gtg gcc ctg 624 Cys Val Val Thr Ile Phe SerIle Ile Leu Leu Ala Val Val Ala Leu 195 200 205 tac gtg cgc atc tac tgcgtg gtc cgc tca agc cac gct gac atg gcc 672 Tyr Val Arg Ile Tyr Cys ValVal Arg Ser Ser His Ala Asp Met Ala 210 215 220 gcc ccg cag acg cta gccctg ctc aag acg gtc acc atc gtg cta ggc 720 Ala Pro Gln Thr Leu Ala LeuLeu Lys Thr Val Thr Ile Val Leu Gly 225 230 235 240 gtc ttt atc gtc tgctgg ctg ccc gcc ttc agc atc ctc ctt ctg gac 768 Val Phe Ile Val Cys TrpLeu Pro Ala Phe Ser Ile Leu Leu Leu Asp 245 250 255 tat gcc tgt ccc gtccac tcc tgc ccg atc ctc tac aaa gcc cac tac 816 Tyr Ala Cys Pro Val HisSer Cys Pro Ile Leu Tyr Lys Ala His Tyr 260 265 270 ctt ttc gcc gtc tccacc ctg aat tcc ctg ctc aac ccc gtc atc tac 864 Leu Phe Ala Val Ser ThrLeu Asn Ser Leu Leu Asn Pro Val Ile Tyr 275 280 285 acg tgg cgc agc cgggac ctg cgg cgg gag gtg ctt cgg ccg ctg cag 912 Thr Trp Arg Ser Arg AspLeu Arg Arg Glu Val Leu Arg Pro Leu Gln 290 295 300 tgc tgg cgg ccg ggggtg ggg gtg caa gga cgg agg cgg ggc ggg acc 960 Cys Trp Arg Pro Gly ValGly Val Gln Gly Arg Arg Arg Gly Gly Thr 305 310 315 320 ccg ggc cac cacctc ctg cca ctc cgc agc tcc agc tcc ctg gag agg 1008 Pro Gly His His LeuLeu Pro Leu Arg Ser Ser Ser Ser Leu Glu Arg 325 330 335 ggc atg cac atgccc acg tca ccc acg ttt ctg gag ggc aac acg gtg 1056 Gly Met His Met ProThr Ser Pro Thr Phe Leu Glu Gly Asn Thr Val 340 345 350 gtc tga 1062 Val4 353 PRT Homo sapiens 4 Met Gly Ser Leu Tyr Ser Glu Tyr Leu Asn Pro AsnLys Val Gln Glu 1 5 10 15 His Tyr Asn Tyr Thr Lys Glu Thr Leu Glu ThrGln Glu Thr Thr Ser 20 25 30 Arg Gln Val Ala Ser Ala Phe Ile Val Ile LeuCys Cys Ala Ile Val 35 40 45 Val Glu Asn Leu Leu Val Leu Ile Ala Val AlaArg Asn Ser Lys Phe 50 55 60 His Ser Ala Met Tyr Leu Phe Leu Gly Asn LeuAla Ala Ser Asp Leu 65 70 75 80 Leu Ala Gly Val Ala Phe Val Ala Asn ThrLeu Leu Ser Gly Ser Val 85 90 95 Thr Leu Arg Leu Thr Pro Val Gln Trp PheAla Arg Glu Gly Ser Ala 100 105 110 Phe Ile Thr Leu Ser Ala Ser Val PheSer Leu Leu Ala Ile Ala Ile 115 120 125 Glu Arg His Val Ala Ile Ala LysVal Lys Leu Tyr Gly Ser Asp Lys 130 135 140 Ser Cys Arg Met Leu Leu LeuIle Gly Ala Ser Trp Leu Ile Ser Leu 145 150 155 160 Val Leu Gly Gly LeuPro Ile Leu Gly Trp Asn Cys Leu Gly His Leu 165 170 175 Glu Ala Cys SerThr Val Leu Pro Leu Tyr Ala Lys His Tyr Val Leu 180 185 190 Cys Val ValThr Ile Phe Ser Ile Ile Leu Leu Ala Val Val Ala Leu 195 200 205 Tyr ValArg Ile Tyr Cys Val Val Arg Ser Ser His Ala Asp Met Ala 210 215 220 AlaPro Gln Thr Leu Ala Leu Leu Lys Thr Val Thr Ile Val Leu Gly 225 230 235240 Val Phe Ile Val Cys Trp Leu Pro Ala Phe Ser Ile Leu Leu Leu Asp 245250 255 Tyr Ala Cys Pro Val His Ser Cys Pro Ile Leu Tyr Lys Ala His Tyr260 265 270 Leu Phe Ala Val Ser Thr Leu Asn Ser Leu Leu Asn Pro Val IleTyr 275 280 285 Thr Trp Arg Ser Arg Asp Leu Arg Arg Glu Val Leu Arg ProLeu Gln 290 295 300 Cys Trp Arg Pro Gly Val Gly Val Gln Gly Arg Arg ArgGly Gly Thr 305 310 315 320 Pro Gly His His Leu Leu Pro Leu Arg Ser SerSer Ser Leu Glu Arg 325 330 335 Gly Met His Met Pro Thr Ser Pro Thr PheLeu Glu Gly Asn Thr Val 340 345 350 Val 5 353 PRT Homo sapiens CONFLICT(272)..(274) Unknown Xaa = Leu or Pro 5 Met Gly Ser Leu Tyr Ser Glu TyrLeu Asn Pro Asn Lys Val Gln Glu 1 5 10 15 His Tyr Asn Tyr Thr Lys GluThr Leu Glu Thr Gln Glu Thr Thr Ser 20 25 30 Arg Gln Val Ala Ser Ala PheIle Val Ile Leu Cys Cys Ala Ile Val 35 40 45 Val Glu Asn Leu Leu Val LeuIle Ala Val Ala Arg Asn Ser Lys Phe 50 55 60 His Ser Ala Met Tyr Leu PheLeu Gly Asn Leu Ala Ala Ser Asp Leu 65 70 75 80 Leu Ala Gly Val Ala PheVal Ala Asn Thr Leu Leu Ser Gly Ser Val 85 90 95 Thr Leu Arg Leu Thr ProVal Gln Trp Phe Ala Arg Glu Gly Ser Ala 100 105 110 Phe Ile Thr Leu SerAla Ser Val Phe Ser Leu Leu Ala Ile Ala Ile 115 120 125 Glu Arg His ValAla Ile Ala Lys Val Lys Leu Tyr Gly Ser Asp Lys 130 135 140 Ser Cys ArgMet Leu Leu Leu Ile Gly Ala Ser Trp Leu Ile Ser Leu 145 150 155 160 ValLeu Gly Gly Leu Pro Ile Leu Gly Trp Asn Cys Leu Gly His Leu 165 170 175Glu Ala Cys Ser Thr Val Leu Pro Leu Tyr Ala Lys His Tyr Val Leu 180 185190 Cys Val Val Thr Ile Phe Ser Ile Ile Leu Leu Ala Ile Val Ala Leu 195200 205 Tyr Val Arg Ile Tyr Cys Val Val Arg Ser Ser His Ala Asp Met Ala210 215 220 Ala Pro Gln Thr Leu Ala Leu Leu Lys Thr Val Thr Ile Val LeuGly 225 230 235 240 Val Phe Ile Val Cys Trp Leu Pro Ala Phe Ser Ile LeuLeu Leu Asp 245 250 255 Tyr Ala Cys Pro Val His Ser Cys Pro Ile Leu TyrLys Ala His Tyr 260 265 270 Xaa Phe Ala Val Ser Thr Leu Asn Ser Leu LeuAsn Pro Val Ile Tyr 275 280 285 Thr Trp Arg Ser Arg Asp Leu Arg Arg GluVal Leu Arg Pro Leu Gln 290 295 300 Cys Trp Arg Pro Gly Val Gly Val GlnGly Arg Arg Arg Gly Gly Thr 305 310 315 320 Pro Gly His His Leu Leu ProLeu Arg Ser Ser Ser Ser Leu Glu Arg 325 330 335 Gly Met His Met Pro ThrSer Pro Thr Phe Leu Glu Gly Asn Thr Val 340 345 350 Val 6 353 PRT Homosapiens 6 Met Gly Ser Leu Tyr Ser Glu Tyr Leu Asn Pro Asn Lys Val GlnGlu 1 5 10 15 His Tyr Asn Tyr Thr Lys Glu Thr Leu Glu Thr Gln Glu ThrThr Ser 20 25 30 Arg Gln Val Ala Ser Ala Phe Ile Val Ile Leu Cys Cys AlaIle Val 35 40 45 Val Glu Asn Leu Leu Val Leu Ile Ala Val Ala Arg Asn SerLys Phe 50 55 60 His Ser Ala Met Tyr Leu Phe Leu Gly Asn Leu Ala Ala SerAsp Leu 65 70 75 80 Leu Ala Gly Val Ala Phe Val Ala Asn Thr Leu Leu SerGly Ser Val 85 90 95 Thr Leu Arg Leu Thr Pro Val Gln Trp Phe Ala Arg GluGly Ser Ala 100 105 110 Phe Ile Thr Leu Ser Ala Ser Val Phe Ser Leu LeuAla Ile Ala Ile 115 120 125 Glu Arg His Val Ala Ile Ala Lys Val Lys LeuTyr Gly Ser Asp Lys 130 135 140 Ser Cys Arg Met Leu Leu Leu Ile Gly AlaSer Trp Leu Ile Ser Leu 145 150 155 160 Val Leu Gly Gly Leu Pro Ile LeuGly Trp Asn Cys Leu Gly His Leu 165 170 175 Glu Ala Cys Ser Thr Val LeuPro Leu Tyr Ala Lys His Tyr Val Leu 180 185 190 Cys Val Val Thr Ile PheSer Ile Ile Leu Leu Ala Val Val Ala Leu 195 200 205 Tyr Val Arg Ile TyrCys Val Val Arg Ser Ser His Ala Asp Met Ala 210 215 220 Ala Pro Gln ThrLeu Ala Leu Leu Lys Thr Val Thr Ile Val Leu Gly 225 230 235 240 Val PheIle Val Cys Trp Leu Pro Ala Phe Ser Ile Leu Leu Leu Asp 245 250 255 TyrAla Cys Pro Val His Ser Cys Pro Ile Leu Tyr Lys Ala His Tyr 260 265 270Leu Phe Ala Val Ser Thr Leu Asn Ser Leu Leu Asn Pro Val Ile Tyr 275 280285 Thr Trp Arg Ser Arg Asp Leu Arg Arg Glu Val Leu Arg Pro Leu Gln 290295 300 Cys Trp Arg Pro Gly Val Gly Val Gln Gly Arg Arg Arg Gly Gly Thr305 310 315 320 Pro Gly His His Leu Leu Pro Leu Arg Ser Ser Ser Ser LeuGlu Arg 325 330 335 Gly Met His Met Pro Thr Ser Pro Thr Phe Leu Glu GlyAsn Thr Val 340 345 350 Val 7 452 DNA human EDG-4 cDNA misc_feature(1)..(7) “n” represents any nucleotide 7 nnnnnnnaaa gccccatggccccagcaggc ctctgagccc caccatgggc agcttgtact 60 cggagtacct gaaccccaacaaggtccagg aacactataa ttataccaag gagacgctgg 120 aaacgcagga gacgacctcccgccaggtgg cctcggcatt catcgtcatc ctctgttgcg 180 ccattgtggt ggaaaaccttctggtgctca ttgcggtggc ccgaaacagc aagttccact 240 cggcaatgta cctgtttctgggcaacctgg ccgcctccga tctactggca ggcgtggcct 300 tcgtagccaa taccttgctctctggctctg tcacgctgag gctgacgcct gtgcagtggt 360 ttgcccggga cggtctgccttcatcacgct ctcggcctct gtcttcagcc tcctggccat 420 cgccattgag cgccacgtggccattgcaaa gg 452 8 452 DNA human EDG-4 cDNA misc_feature (1)..(7) “n”represents any nucleotide 8 nnnnnnnaaa gccccatggc cccagcaggc ctctgagccccaccatgggc agcttgtact 60 cggagtacct gaaccccaac aaggtccagg aacactataattataccaag gagacgctgg 120 aaacgcagga gacgacctcc cgccaggtgg cctcggccttcatcgtcatc ctctgttgcg 180 ccattgtggt ggaaaacctt ctggtgctca ttgcggtggcccgaaacagc aagttccact 240 cggcaatgta cctgtttctg ggcaacctgg ccgcctccgatctactggca ggcgtggcct 300 tcgtagccaa taccttgctc tctggctctg tcacgctgaggctgacgcct gtgcagtggt 360 ttgcccggga cnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn 452 9 451DNA human EDG-4 cDNA misc_feature (370)..(451) “n” represents anynucleotide 9 agttctgaaa gccccatggc cccagcaggc ctctgagccc caccatgggcagcttgtact 60 cggagtacct gaaccccaac aaggtccagg aacactataa ttataccaaggagacgctgg 120 aaacgcagga gacgacctcc cgccaggtgg gctcggcctt catcgtcatcctctgttgcg 180 ccattgtggt ggaaaacctt ctggtgctca ttgcggtggc ccgaaacagcaagttccact 240 cggcaatgta cctgtttctg ggcaacctgg ccgcctccga tctactggcaggcgtggctt 300 cgtagccaat accttgctct ctggctctgt cacgctgagg ctgacgcctgtgcagtggtt 360 tgcccgggan nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn n 451 10 353 PRT humanEDG-4 polypeptide misc_feature (273)..(273) “Xaa” represents any aminoacid 10 Met Gly Ser Leu Tyr Ser Glu Tyr Leu Asn Pro Asn Lys Val Gln Glu1 5 10 15 His Tyr Asn Tyr Thr Lys Glu Thr Leu Glu Thr Gln Glu Thr ThrSer 20 25 30 Arg Gln Val Ala Ser Ala Phe Ile Val Ile Leu Cys Cys Ala IleVal 35 40 45 Val Glu Asn Leu Leu Val Leu Ile Ala Val Ala Arg Asn Ser LysPhe 50 55 60 His Ser Ala Met Tyr Leu Phe Leu Gly Asn Leu Ala Ala Ser AspLeu 65 70 75 80 Leu Ala Gly Val Ala Phe Val Ala Asn Thr Leu Leu Ser GlySer Val 85 90 95 Thr Leu Arg Leu Thr Pro Val Gln Trp Phe Ala Arg Glu GlySer Ala 100 105 110 Phe Ile Thr Leu Ser Ala Ser Val Phe Ser Leu Leu AlaIle Ala Ile 115 120 125 Glu Arg His Val Ala Ile Ala Lys Val Lys Leu TyrGly Ser Asp Lys 130 135 140 Ser Cys Arg Met Leu Leu Leu Ile Gly Ala SerTrp Leu Ile Ser Leu 145 150 155 160 Val Leu Gly Gly Leu Pro Ile Leu GlyTrp Asn Cys Leu Gly His Leu 165 170 175 Glu Ala Cys Ser Thr Val Leu ProLeu Tyr Ala Lys His Tyr Val Leu 180 185 190 Cys Val Val Thr Ile Phe SerIle Ile Leu Leu Ala Ile Val Ala Leu 195 200 205 Tyr Val Arg Ile Tyr CysVal Val Arg Ser Ser His Ala Asp Met Ala 210 215 220 Ala Pro Gln Thr LeuAla Leu Leu Lys Thr Val Thr Ile Val Leu Gly 225 230 235 240 Val Phe IleVal Cys Trp Leu Pro Ala Phe Ser Ile Leu Leu Leu Asp 245 250 255 Tyr AlaCys Pro Val His Ser Cys Pro Ile Leu Tyr Lys Ala His Tyr 260 265 270 XaaPhe Ala Val Ser Thr Leu Asn Ser Leu Leu Asn Pro Val Ile Tyr 275 280 285Thr Trp Arg Ser Arg Asp Leu Arg Arg Glu Val Leu Arg Pro Leu Gln 290 295300 Cys Trp Arg Pro Gly Val Gly Val Gln Gly Arg Arg Arg Gly Gly Thr 305310 315 320 Pro Gly His His Leu Leu Pro Leu Arg Ser Ser Ser Ser Leu GluArg 325 330 335 Gly Met His Met Pro Thr Ser Pro Thr Phe Leu Glu Gly AsnThr Val 340 345 350 Val 11 352 PRT rat EDG-4 polypeptide 11 Met Gly GlyLeu Tyr Ser Glu Tyr Leu Asn Pro Glu Lys Val Gln Glu 1 5 10 15 His TyrAsn Tyr Thr Lys Glu Thr Leu Asp Met Gln Glu Thr Pro Ser 20 25 30 Arg LysVal Ala Ser Ala Phe Ile Ile Ile Leu Cys Cys Ala Ile Val 35 40 45 Val GluAsn Leu Leu Val Leu Ile Ala Val Ala Arg Asn Ser Lys Phe 50 55 60 His SerAla Met Tyr Leu Phe Leu Gly Asn Leu Ala Ala Ser Asp Leu 65 70 75 80 LeuAla Gly Val Ala Phe Val Ala Asn Thr Leu Leu Ser Gly Pro Val 85 90 95 ThrLeu Ser Leu Thr Pro Leu Gln Trp Phe Ala Arg Glu Gly Ser Ala 100 105 110Phe Ile Thr Leu Ser Ala Ser Val Phe Ser Leu Leu Ala Ile Ala Ile 115 120125 Glu Arg Gln Val Ala Ile Ala Lys Val Lys Leu Tyr Gly Ser Asp Lys 130135 140 Ser Cys Arg Met Leu Met Leu Ile Gly Ala Ser Trp Leu Ile Ser Leu145 150 155 160 Ile Leu Gly Gly Leu Pro Ile Leu Gly Trp Asn Cys Leu AspHis Leu 165 170 175 Glu Ala Cys Ser Thr Val Leu Pro Leu Tyr Ala Lys HisTyr Val Leu 180 185 190 Cys Val Val Thr Ile Phe Ser Val Ile Leu Leu AlaIle Val Ala Leu 195 200 205 Tyr Val Arg Ile Tyr Phe Val Val Arg Ser SerHis Ala Asp Val Ala 210 215 220 Gly Pro Gln Thr Leu Ala Leu Leu Lys ThrVal Thr Ile Val Leu Gly 225 230 235 240 Val Phe Ile Ile Cys Trp Leu ProAla Phe Ser Ile Leu Leu Leu Asp 245 250 255 Ser Thr Cys Pro Val Arg AlaCys Pro Val Leu Tyr Lys Ala His Tyr 260 265 270 Phe Phe Ala Phe Ala ThrLeu Asn Ser Leu Leu Asn Pro Val Ile Tyr 275 280 285 Thr Trp Arg Ser ArgAsp Leu Arg Arg Glu Val Leu Arg Pro Leu Leu 290 295 300 Cys Trp Arg GlnGly Lys Gly Ala Thr Gly Arg Arg Gly Gly Asn Pro 305 310 315 320 Gly HisArg Leu Leu Pro Leu Arg Ser Ser Ser Ser Leu Glu Arg Gly 325 330 335 LeuHis Met Pro Thr Ser Pro Thr Phe Leu Glu Gly Asn Thr Val Val 340 345 35012 353 PRT human EDG-4 #36 12 Met Gly Ser Leu Tyr Ser Glu Tyr Leu AsnPro Asn Lys Val Gln Glu 1 5 10 15 His Tyr Asn Tyr Thr Lys Glu Thr LeuGlu Thr Gln Glu Thr Thr Ser 20 25 30 Arg Gln Val Ala Ser Ala Phe Ile ValIle Leu Cys Cys Ala Ile Val 35 40 45 Val Glu Asn Leu Leu Val Leu Ile AlaVal Ala Arg Asn Ser Lys Phe 50 55 60 His Ser Ala Met Tyr Leu Phe Leu GlyAsn Leu Ala Ala Ser Asp Leu 65 70 75 80 Leu Ala Gly Val Ala Phe Val AlaAsn Thr Leu Leu Ser Gly Ser Val 85 90 95 Thr Leu Arg Leu Thr Pro Val GlnTrp Phe Ala Arg Glu Gly Ser Ala 100 105 110 Phe Ile Thr Leu Ser Ala SerVal Phe Ser Leu Leu Ala Ile Ala Ile 115 120 125 Glu Arg His Val Ala IleAla Lys Val Lys Leu Tyr Gly Ser Asp Lys 130 135 140 Ser Cys Arg Met LeuLeu Leu Ile Gly Ala Ser Trp Leu Ile Ser Leu 145 150 155 160 Val Leu GlyGly Leu Pro Ile Leu Gly Trp Asn Cys Leu Gly His Leu 165 170 175 Glu AlaCys Ser Thr Val Leu Pro Leu Tyr Ala Lys His Tyr Val Leu 180 185 190 CysVal Val Thr Ile Phe Ser Ile Ile Leu Leu Ala Val Val Ala Leu 195 200 205Tyr Val Arg Ile Tyr Cys Val Val Arg Ser Ser His Ala Asp Met Ala 210 215220 Ala Pro Gln Thr Leu Ala Leu Leu Lys Thr Val Thr Ile Val Leu Gly 225230 235 240 Val Phe Ile Val Cys Trp Leu Pro Ala Phe Ser Ile Leu Leu LeuAsp 245 250 255 Tyr Ala Cys Pro Val His Ser Cys Pro Ile Leu Tyr Lys AlaHis Tyr 260 265 270 Leu Phe Ala Val Ser Thr Leu Asn Ser Leu Leu Asn ProVal Ile Tyr 275 280 285 Thr Trp Arg Ser Arg Asp Leu Arg Arg Glu Val LeuArg Pro Leu Gln 290 295 300 Cys Trp Arg Pro Gly Val Gly Val Gln Gly ArgArg Arg Gly Gly Thr 305 310 315 320 Pro Gly His His Leu Leu Pro Leu ArgSer Ser Ser Ser Leu Glu Arg 325 330 335 Gly Met His Met Pro Thr Ser ProThr Phe Leu Glu Gly Asn Thr Val 340 345 350 Val 13 352 PRT human EDG-6receptor misc_feature (352)..(352) “Xaa” represents any amino acid 13Met Val Ile Met Gly Gln Cys Tyr Tyr Asn Glu Thr Ile Gly Phe Phe 1 5 1015 Tyr Asn Asn Ser Gly Lys Glu Leu Ser Ser His Trp Arg Pro Lys Asp 20 2530 Val Val Val Val Ala Leu Gly Leu Thr Val Ser Val Leu Val Leu Leu 35 4045 Thr Asn Leu Leu Val Ile Ala Ala Ile Ala Ser Asn Arg Arg Phe His 50 5560 Gln Pro Ile Tyr Tyr Leu Leu Gly Asn Leu Ala Ala Ala Asp Leu Phe 65 7075 80 Ala Gly Val Ala Tyr Leu Phe Leu Met Phe His Thr Gly Pro Arg Thr 8590 95 Ala Arg Leu Ser Leu Glu Gly Trp Phe Leu Arg Gln Gly Leu Leu Asp100 105 110 Thr Ser Leu Thr Ala Ser Val Ala Thr Leu Leu Ala Ile Ala ValGlu 115 120 125 Arg His Arg Ser Val Met Ala Val Gln Leu His Ser Arg LeuPro Arg 130 135 140 Gly Arg Val Val Met Leu Ile Val Gly Val Trp Val AlaAla Leu Gly 145 150 155 160 Leu Gly Leu Leu Pro Ala His Ser Trp His CysLeu Cys Ala Leu Asp 165 170 175 Arg Cys Ser Arg Met Ala Pro Leu Leu SerArg Ser Tyr Leu Ala Val 180 185 190 Trp Ala Leu Ser Ser Leu Leu Val PheLeu Leu Met Val Ala Val Tyr 195 200 205 Thr Arg Ile Phe Phe Tyr Val ArgArg Arg Val Gln Arg Met Ala Glu 210 215 220 His Val Ser Cys His Pro ArgTyr Arg Glu Thr Thr Leu Ser Leu Val 225 230 235 240 Lys Thr Val Val IleIle Leu Gly Ala Phe Val Val Cys Trp Thr Pro 245 250 255 Gly Gln Val ValLeu Leu Leu Asp Gly Leu Gly Cys Glu Ser Cys Asn 260 265 270 Val Leu AlaVal Glu Lys Tyr Phe Leu Leu Leu Ala Glu Ala Asn Ser 275 280 285 Leu ValAsn Ala Ala Val Tyr Ser Cys Arg Asp Ala Glu Met Arg Arg 290 295 300 ThrPhe Arg Arg Leu Leu Cys Cys Ala Cys Leu Arg Gln Ser Thr Arg 305 310 315320 Glu Ser Val His Tyr Thr Ser Ser Ala Gln Gly Gly Ala Ser Thr Arg 325330 335 Ile Met Leu Pro Glu Asn Gly His Pro Leu Met Asp Ser Thr Leu Xaa340 345 350 14 1056 DNA human EDG-6 receptor 14 atggtcatca tgggccagtgctactacaac gagaccatcg gcttcttcta taacaacagt 60 ggcaaagagc tcagctcccactggcggccc aaggatgtgg tcgtggtggc actggggctg 120 accgtcagcg tgctggtgctgctgaccaat ctgctggtca tagcagccat cgcctccaac 180 cgccgcttcc accagcccatctactacctg ctcggcaatc tggccgcggc tgacctcttc 240 gcgggcgtgg cctacctcttcctcatgttc cacactggtc cccgcacagc ccgactttca 300 cttgagggct ggttcctgcggcagggcttg ctggacacaa gcctcactgc gtcggtggcc 360 acactgctgg ccatcgccgtggagcggcac cgcagtgtga tggccgtgca gctgcacagc 420 cgcctgcccc gtggccgcgtggtcatgctc attgtgggcg tgtgggtggc tgccctgggc 480 ctggggctgc tgcctgcccactcctggcac tgcctctgtg ccctggaccg ctgctcacgc 540 atggcacccc tgctcagccgctcctatttg gccgtctggg ctctgtcgag cctgcttgtc 600 ttcctgctca tggtggctgtgtacacccgc attttcttct acgtgcggcg gcgagtgcag 660 cgcatggcag agcatgtcagctgccacccc cgctaccgag agaccacgct cagcctggtc 720 aagactgttg tcatcatcctgggggcgttc gtggtctgct ggacaccagg ccaggtggta 780 ctgctcctgg atggtttaggctgtgagtcc tgcaatgtcc tggctgtaga aaagtacttc 840 ctactgctgg ccgaggccaactcactggtc aatgctgctg tgtactcttg ccgagatgct 900 gagatgcgcc gcaccttccgccgccttctc tgctgcgcgt gcctccgcca gtccacccgc 960 gagtctgtcc actatacatcctctgcccag ggaggtgcca gcactcgcat catgcttccc 1020 gagaacggcc acccactgatggactccacc ctttag 1056

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. An isolatedpolynucleotide that codes for an endothelial differentiation gene (EDG)receptor, said EDG receptor comprising the amino acid sequence, selectedfrom the group consisting of: (a) the amino acid sequence comprising SEQID NO:2 and (b) the amino acid sequence comprising SEQ ID NO:4.
 2. Anisolated polynucleotide according to claim 1 that codes for an EDGreceptor of the amino acid sequence comprising the amino acids of SEQ IDNO:
 2. 3. An isolated polynucleotide according to claim 1 that codes foran EDG receptor of the amino acid sequence comprising the amino acids ofSEQ ID NO):
 4. 4. An isolated polynucleotide according to claim 1comprising the nucleotide sequence comprising nucleotides 38-1099 of SEQID NO:
 1. 5. An isolated polynucleotide according to claim 1 comprisingthe nucleotide sequence of SEQ ID NO:
 3. 6. A vector comprising apolynucleotide according to claim
 1. 7. A vector according to claim 6comprising a polynucleotide that codes for an EDG receptor of the aminoacid sequence comprising the amino acids of SEQ ID NO:
 2. 8. A vectoraccording to claim 6 comprising a polynucleotide that codes for an EDGreceptor of the amino acid sequence comprising the amino acids of SEQ IDNO:
 4. 9. A cell that has been genetically engineered to produce an EDGreceptor wherein said cell has incorporated expressibly therein apolynucleotide as defined in claim
 1. 10. A cell according to claim 9wherein said cell has incorporated expressibly therein a polynucleotidethat codes for an EDG receptor of the amino acid sequence comprising theamino acids of SEQ ID NO:
 2. 11. A cell according to claim 9 whereinsaid cell has incorporated expressibly therein a polynucleotide thatcodes for EDG receptor of the amino acid sequence comprising the aminoacids of SEQ ID NO:
 4. 12. A membrane preparation obtained from a cellas defined in claim
 9. 13. A membrane preparation obtained from a cellas defined in claim
 10. 14. A membrane preparation obtained from a cellas defined in claim
 11. 15. An isolated EDG receptor comprising theamino acid sequence of SEQ ID NO:
 2. 16. An isolated EDG receptorcomprising the amino acid sequence of SEQ ID NO: 4.